Anti-TEM1 antibodies and uses thereof

ABSTRACT

The invention relates to Anti-TEM 1 antibodies or antigen-binding fragments thereof, yeast libraries comprising the same, and prophylactic, diagnostic, and therapeutic methods using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/411,938, filed on Jan. 20, 2017 which is a Divisional of U.S. patentapplication Ser. No. 13/508,925, filed on Aug. 1, 2012 which is aNational Phase Application of PCT International Application No.PCT/US10/56477, International Filing Date Nov. 12, 2010, claimingpriority to U.S. Provisional Patent Application 61/260,286, filed Nov.11, 2009, each of which is incorporated by reference herein in itsentirety.

GOVERNMENT INTEREST

The work described in this patent application was supported, in part, bythe Institute for the Translational Medicine and Therapeutics(CA016520/NIH) (NS), and the National Heart, Lung, and Blood Institute(P50-HL81012) (DLS). The Unites States government may have certainrights in this patent application.

FIELD OF THE INVENTION

The invention relates to an antibody or antigen-binding fragment thatbinds specifically to an endosialin tumor endothelial marker 1 (TEM1),and prophylactic, diagnostic, and therapeutic methods using the same.

BACKGROUND OF THE INVENTION

Isolation of antigen-specific antibodies has been achieved through avariety of methods, including screening of phage-display recombinantantibody (scFv) libraries. Yeast-display recently emerged as anefficient alternative strategy for scFv identification that offersseveral advantages over prokaryotic systems, including superior samplingof the immune antibody repertoire; post-translational modifications(glycosylation) due to the eukaryotic expression; faster and morecontrolled flow cytometry-based selection compared to solid phasepanning; and absence of growth bias, as recombinant proteins aredisplayed at the yeast cell surface only during the induction step inthe presence of galactose. Yet, previously reported yeast libraries havebeen severely limited in size, with typically less than 1×10⁵transformants per microgram of DNA for commonly used strains, resultingin insufficient diversity and potential for yielding high affinityantibodies. Additionally, with existing methods, transfer of scFv fromdisplayed to secreted forms has often resulted in loss of antigenspecificity and/or affinity, requiring additional time-consuming andcostly steps, including in vitro maturation of scFv sequence and/orrecloning of scFv fused to immunoglobulin (Ig) constant regions. Themechanisms underlying loss of scFv function include changes in scFvconformation and post-translational modification due to differentexpression systems for displayed and secreted forms.

It was hypothesized that the use of electroporation combined with buffermodifications could remove obstacles contributed by poor yeasttransformation efficiency. In addition, we hypothesized that only oneexpression system (Saccharomyces cerevisiae) for both scFv display andsecretion could eliminate changes in scFv post-translationalmodifications, while keeping the advantages of an eukaryotic system forthe expression of high-affinity antibodies. It was also hypothesizedthat if both displayed and secreted scFv were modified only at theN-terminus, which binds to the yeast surface or to secondary reagents,respectively, conformational changes would be minimized during the shiftfrom displayed to secreted forms. To test this hypotheses, a previouslygenerated M13 bacteriophage display human scFv library was transferredthrough homologous recombination into our novel vector pAGA2 foryeast-display. This yielded a 1×10⁹-member yeast scFv display library,which was then screened in two steps using two novel complementary yeastsystems. The first was engineered to permit scFv surface expression as afusion with an Aga2 protein to N-termini for convenient high-throughputscreening by flow sorting. The second was engineered to permit rapidtransformation into yeast-secreted soluble scFvs fused to N-termini toan IgA hinge and an enzymatically biotinylatable site for in vitro andin vivo validation. Such scFv are called “biobodies” after targetedbiotinylation by yeast mating. As proof of principle, we used this novelplatform to screen for scFv against the tumor marker endosialin/tumorendothelial marker 1 (TEM1) or CD248, an attractive target forantibody-based tumor diagnosis and therapy. Several high-affinityTEM1-specific yeast-secreted scFvs and biobodies were isolated andcharacterized for binding to human and murine TEM1 in vitro and in vivo.The highest affinity anti-TEM1 biobody-78 (Kd 4 nM) was able to bindboth murine and human TEM1 and selectively targeted TEM1-expressingtumor cells in a novel in vivo mouse model of orthotopic ovarian cancer.This streamlined approach for rapid identification of high affinityreagents suitable for in vivo use paves the way for the high throughputdevelopment of novel antigen-targeted theranostics.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides an isolated antibody orantigen-binding fragment, whereby said antibody or antigen-bindingfragment is, in another embodiment, specific for both the mouse andhuman form of an endosialin tumor endothelial marker 1 (TEM1).

In another embodiment, the invention provides a method of treating atumor in a subject comprising the step of contacting said tumor cellwith said antibody or antigen-binding fragment thereof of that isoperably linked to a biologically active agent, wherein said agent is atoxin, a radioisotope, a nanoparticle or a bio-active peptide.

In another embodiment, the invention provides a method of treatingangiogenesis of a solid tumor in a subject, said method comprising thestep of contacting a pericyte of said solid tumor with said antibody orantigen-binding fragment thereof of is operably linked to a biologicallyactive agent, wherein said agent is a toxin, a radioisotope, ananoparticle or a bio-active peptide.

In another embodiment, the invention provides a method of diagnosing thepresence of a tumor or a cancer growth in a subject, said methodcomprising sampling a tissue sample isolated from said subject with acomposition comprising said antibody or antigen-binding fragment,whereby specific binding of said antibody or antigen-binding fragment tosaid tissue sample is indicative of the presence of a tumor or cancergrowth in said subject.

In another embodiment, the invention provides a method of imaging aTEM1-containing tumor, said method comprising the step of applying saidantibody or antigen-binding fragment operably linked to a secondaryreagent; whereby said secondary reagent can be visualized once saidantibody or antigen-binding fragment has bound its target TEM1.

Other features and advantages of the present invention will becomeapparent from the following detailed description examples and figures.It should be understood, however, that the detailed description and thespecific examples while indicating preferred embodiments of theinvention are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. Companion vectors for yeast-display and yeast-secreted scFvexpression. a-b: Overall structures of yeast-display (a) andyeast-secreted scFv (b). c: pAGA2 vector for yeast-display. The shuttlevector p414 GAL1 that allows galactose-inducible expression in presenceof uracil was modified to include Nhe1, EcoR1 and Xho1 restrictionenzyme sites matching the cloning sites of its companion vector p416BCCP (d). The multiple cloning site (MCS) was engineered by inserting aNhe1 site after the FLAG tag and (G₄S)₃ linker sequence; an EcoR1 site,also part of a stop codon that is removed when cDNA are inserted inframe in the cloning site; and a Xho1 site, inserted directly before thec-myc tag, out of frame with the FLAG tag, to insure that both tagswould be expressed only in presence of correctly inserted cDNA. d:p416-BCCP for yeast-secretion. The shuttle vector p416 GAL1 that allowsfor galactose-inducible secretion in the presence of tryptophane waslinearized by BamH1 and Xho1 and co-transformed in yeast with a purifiedPCR product encoding alpha preproleader and RK endopeptidase sequencesfollowed by biotin accepting site (BCCP), IgA hinge, FLAG tag, (G₄S)₃linker, cloning site with stop codon, and V5-HIS tags.

FIG. 2. Optimization of yeast transformations a-b Chemicaltransformations. Calibration of chemical transformation: a. EBY100 cellsin log phase were treated by five different conditions described inTable 1. The number of transformants ×10⁴ per μg of plasmid DNA isplotted on the Y axis. b. Chemical transformation of EBY100 (black bars)vs. YVH10 (grey bars) with different concentrations of intact DNAplasmids (1 μg, 5 μg and 10 μg, as indicated) or of combinations oflinearized plasmid and insert for homologous recombination (1 μg, 2.5 μgand 5 μg of linearized vector combined with 3-fold molar excess of scFvfragments, as indicated). The number of transformants ×10⁶ is plotted onthe Y axis. c-d Electroporations. All experiments were performed in 0.2cm cuvettes, in 50 ml volume, with settings of 1.5 kV, 25 mF and 200Ω.c. Calibrations: EBY100 cells in log phase were treated with fifteendifferent conditions (a-o) as described in Table 2. The number oftransformants ×10⁶ per transformation with 1 μg of plasmid DNA isplotted on the Y axis. d. Electroporation of EBY100 in condition G, withdifferent concentrations of linearized vector (white bars) or intact DNAplasmid (grey bars) or combination of linearized plasmid and insert(black bars) for homologous recombinations (as indicated). The number oftransformants per transformation is plotted on the Y axis.

FIG. 3: Calibration of YVH10 electroporation conditions. YVH10 wereelectroporated with increasing amount of intact plasmid (white bars) orlinearized plasmid and insert (black bars) to promote homologousrecombinations. Electroporations were performed in 100 ul volume in 0.2cm cuvette; the apparatus setting was 1.5 kV, 25 μF, and 200Ω.

FIG. 4: Flow cytometry analysis of yeast-display scFv library.Yeast-display scFv were detected by anti-c-myc mAb followed up by488-labeled anti-mIg.

FIG. 5: Germline immunoglobulin gene usage and predicted amino acidsequences of anti-TEM1 scFv a. Anti-TEM1 heavy chain variable regions.The number of nucleotide differences from germline V_(H) is tabulated tothe right of each sequence. In general, D segments showed very poorhomology with known D genes so mutations were not scored in theseregions. FR (framework region) and CDR (complementarily determiningregion) designations as per Kabat. b. Anti-TEM1 light chain variableregions. The number of nucleotide differences from germline V_(L) istabulated to the right of each sequence.

FIG. 6: Measurement of scFv-78 affinity by ELISA. Kd was calculated bythe equation Kd=2[Ab′]t−[Ab]t, where [Ab′]t refers to the scFvconcentration at OD₅₀ for the half concentration rhTEM1 coated wellswhile [Ab]t refers to the scFv concentration at OD50 for oneconcentration rhTEM1-GST coated wells. The calculated Kd for scFv-78 was5.8 nM using antigen concentrations of 0.4 μg/ml (diamonds) and 0.2μg/ml (squares), 4.5 nM using antigen concentrations of 0.2 μg/ml and0.1 μg/ml (open triangles), or 2.8 nM using antigen concentration of 0.1μg/ml and 0.05 μg/ml (open circles). ELISAs were performed in duplicatein two independent experiments. Lines are fitted using theantibody-antigen reaction equation.

FIG. 7: Measurement of scFv-132; scFv-133; scFv-131 and scFv-137affinities by ELISA. Kd was calculated by the equation Kd=2[Ab′]t−[Ab]t,where [Ab′]t refers to the scFv concentration at OD50 for the halfconcentration rhTEM1 coated wells while [Ab]t refers to the scFvconcentration at OD50 for one concentration rhTEM1-coated wells. Thecalculated Kd's for each scFv are as follows: (a), 148 nM, scFv-132,(b), 218 nM, scFv-133, (c), 628 nM, scFv-131, and (d), 4.4 μM, scFv-137,using 2 μg/ml (squares) or 1 μg/ml (triangle) of rhTEM1. ELISAs wereperformed in duplicate in two independent experiments. Lines are fittedusing the antibody-antigen reaction equation.

FIG. 8: Characterization of anti-TEM1 scFv binding. Wild type (a-f) andhTEM1-transfected (g-1) microvascular endothelial cells of murinepancreatic origin (Mile-Sven, MS1 cells, American Type CultureCollection, Manassas, Va.) were incubated with five different anti-TEM1scFv (as indicated) at 10 nM for scFv-78 and at 1 μM for scFv-132,scFv-133, scFv-131, scFv-137, and an irrelevant control scFv. ScFvbinding to cell surface was detected with APC-labeled anti-V5 (solidblack line). Blocking conditions were performed in presence of 20 nM ofrhTEM1-GST (small dotted line) or 100 nM of rGST control protein (dashedline) for scFv78 and 1 μM of rhTEM1-GST (small dotted line) or 1 μM ofrGST control protein (dashed line) for scFv-131, -132, -133 and -137. Asa negative control, cells were incubated with APC anti-V5 mAb only (greyline). m-s: Targeted biotinylated ScFv-78 (biobody-78) was incubatedwith TEM1-endogenous expresser embryonic kidney 293 cells (HEK293) (m);wild type heart endothelial mouse cells (H5V) (n); hTEM1-transfected H5Vcells (o); wild type human ovarian cancer cells (SKOV3) (p);hTEM1-transfected SKOV3 cells (q); mouse TEM1-endogenous expresserendothelial cells (2H11) (r), and mouse TEM1-endogenous expresserovarian cancer cells (MOV-1) (s). Biobody-78 binding was detected by 30nM of APC-labeled streptavidin (black line). As a negative control,cells were incubated with APC-labeled streptavidin only (grey line).

FIG. 9: TEM1 transcript expression levels in murine and human celllines. a. Human endogenous and transduced expression levels of TEM1 weremeasured in wild type or hTEM-1-transduced murine endothelial cell linesMS1 and H5V cell lines, in human fibroblast cell line HEK293T and inhuman ovarian cancer cell line SKOV3 by qRT PCR, as indicated. b. Murineendogenous TEM1 transcript expression levels were measured in murineendothelial cell lines MS1, HSV, 2H11 and ovarian cancer cell line MOV1,as indicated.

FIG. 10: Biodistribution of anti-TEM1 biobodies. Mice were firstimplanted in the left bursa with MOV1 cells, IV-injected three weekslater with anti-TEM1 biobody-78 (a-j) or biobody-137 (k-o), andsacrificed 24 (a-e) or 48 hours (f-o) later. Bindings of anti-TEM1biobodies to the left ovary (a,f,k), right ovary (b,g,l), spleen(c,h,m), liver (d, i, n) and kidney (e, j, o) were assessed by confocalmicroscopy (63×) by staining with 1 μg/ml of rhodamine-conjugatedstreptavidin (2). Presence of tumor cells was detected with 2 μg/ml ofanti-SV40 mAb followed by 1 μg/ml of alexa-488 goat anti-mouse IgG2a(1). DAPI staining to visualized nuclei (3). (4) Merged images of (1),(2) and (3).

FIG. 11: scFv78-biob-SA-PE complex was prepared by pre-incubation ofscFv78-biob and SA-PE for 30 min at 4 C with the ration of 4:1 and theconcentration of SA-PE at 50 nM. Peptide displaying yeasts wereincubated with scFv78-biob-SA-PE for 1 h at 4 C and scFv78-biob bindingwas evaluated by FACS.

FIG. 12: Anti-TEM1 recombinant antibody internalizes in TEM1-expressercells. MS1 endothelial cells, wild type (A,E,F) or transduced to expressTEM1 (MS1-TEM1, B-D,G-H) were fixed, permeabilized, and incubated for 15hours at 37° C. with anti-TEM1 scFv78 (A,D) or Biobody 78 (Bb78, F,H).Cells were then washed and incubated with alexa647-labeled anti-V5antibody (A-D) or with dylight649-labeled beads (E-H). As negativecontrols, cells were incubated in medium only (B), or in mediumsupplemented with V5-tagged B7H4 protein (C) or BSA (E, G).

DETAILED DESCRIPTION OF THE INVENTION

This invention relates in one embodiment to isolated antibodies orantigen-binding fragments thereof that selectively bind the same humanand mouse target, methods of treatment comprising administering saidantibodies or antigen-binding fragments, and yeast libraries comprisingsaid antibodies or antigen-binding fragments in display or secretableforms. In one embodiment, provided herein is an isolated antibody orantigen-binding fragment that is specific for both the mouse and humanform of an endosialin tumor endothelial marker 1 (TEM1). In anotherembodiment, the antibody or antigen-binding fragment binds the TEM1epitope exemplified herein (see Examples). In another embodiment, theantibody or antigen-binding fragment binds the TEM1 epitope sequence setforth in SEQ ID NO: 40, provided herein below.

In some embodiments, the term “epitope” refers to a region of theantigen that binds to the antibody. It is the region of an antigenrecognized by a first antibody wherein the binding of the first antibodyto the region prevents binding of a second antibody or other bivalentmolecule to the region. The region encompasses a particular coresequence or sequences selectively recognized by a class of antibodies.In general, epitopes are comprised by local surface structures that canbe formed by contiguous or noncontiguous amino acid sequences.

In another embodiment, the term “Selectively recognizes”, “selectivelybind” or “selectively recognized” means that binding of the antibody orother bivalent molecule to an epitope is at least 2-fold greater,preferably 2-5 fold greater, and most preferably more than 5-foldgreater than the binding of the bivalent molecule to an unrelatedepitope or than the binding of an unrelated bivalent molecule to theepitope, as determined by techniques known in the art and describedherein, such as, for example, ELISA and cold displacement assays. Insome embodiments, the term “antibody” refers to the structure thatconstitutes the natural biological form of an antibody. In most mammals,including humans, and mice, this form is a tetramer and consists of twoidentical pairs of two immunoglobulin chains, each pair having one lightand one heavy chain, each light chain comprising immunoglobulin domainsV_(L) and C_(L), and each heavy chain comprising immunoglobulin domainsV_(H), Cγ1, Cγ2, and Cγ3. In each pair, the light and heavy chainvariable regions (V_(L) and V_(H)) are together responsible for bindingto an antigen, and the constant regions (C_(L), Cγ1, Cγ2, and Cγ3,particularly Cγ2, and Cγ3) are responsible for antibody effectorfunctions. In some mammals, for example in camels and llamas,full-length antibodies may consist of only two heavy chains, each heavychain comprising immunoglobulin domains V_(H), Cγ2, and Cγ3. By“immunoglobulin (Ig)” herein is meant a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genesImmunoglobulins include but are not limited to antibodiesImmunoglobulins may have a number of structural forms, including but notlimited to full-length antibodies, antibody fragments, and individualimmunoglobulin domains including but not limited to V_(H), Cγ1, Cγ2,Cγ3, V_(L), and C_(L).

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes”.There are five-major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called alpha, delta, epsilon, gamma, and mu, respectively. Thesubunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

In some embodiments, the term “antibody” or “antigen-binding fragment”respectively refer to intact molecules as well as functional fragmentsthereof, such as Fab, a scFv-Fc bivalent molecule, F(ab′)₂, and Fv thatare capable of specifically interacting with a desired target. In someembodiments, the antigen-binding fragments comprise:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, which can be produced by digestion ofwhole antibody with the enzyme papain to yield an intact light chain anda portion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule that can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)2 is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains; and

(5) Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

(6) scFv-Fc, is produced in one embodiment, by fusing single-chain Fv(scFv) with a hinge region from an immunoglobulin (Ig) such as an IgG,and Fc regions.

In one embodiment, the antibody provided herein is a monoclonalantibody. In another embodiment, the antigen-binding fragment providedherein is a single chain Fv (scFv), a diabody, a tandem scFv, a scFv-Fcbivalent molecule, an Fab, Fab′, Fv, or F(ab′)₂.

In one embodiment, the term “Bivalent molecule” or “BV” refers to amolecule capable of binding to two separate targets at the same time.The bivalent molecule is not limited to having two and only two bindingdomains and can be a polyvalent molecule or a molecule comprised oflinked monovalent molecules. The binding domains of the bivalentmolecule can selectively recognize the same epitope or differentepitopes located on the same target or located on a target thatoriginates from different species. The binding domains can be linked inany of a number of ways including, but not limited to, disulfide bonds,peptide bridging, amide bonds, and other natural or synthetic linkagesknown in the art (Spatola et al., “Chemistry and Biochemistry of AminoAcids, Peptides and Proteins,” B. Weinstein, eds., Marcel Dekker, NewYork, p. 267 (1983) (general review); Morley, J. S., “Trends Pharm Sci”(1980) pp. 463-468 (general review); Hudson et al., Int J Pept Prot Res(1979) 14, 177-185; Spatola et al., Life Sci (1986) 38, 1243-1249; Hann,M. M., J Chem Soc Perkin Trans I (1982) 307-314; Almquist et al., J MedChem (1980) 23, 1392-1398; Jennings-White et al., Tetrahedron Lett(1982) 23, 2533; Szelke et al., European Application EP 45665; ChemicalAbstracts 97, 39405 (1982); Holladay, et al., Tetrahedron Lett (1983)24, 4401-4404; and Hruby, V. J., Life Sci (1982) 31, 189-199).

In another embodiment, the antigen-binding fragment thereof is highaffinity anti-TEM1 scFv-78. In another embodiment, the antigen-bindingfragment thereof is low affinity anti-TEM1 scFv-137. In anotherembodiment scFv78 specifically binds the highly conserved fragment ofthe mouse and human TEM1 proteins comprising amino acids 324 to 390where and in other embodiments, said fragment is the T6 fragment of theTEM1 protein located in the middle of the extracellular domain.

In one embodiment, the term “binds” or “binding” or grammaticalequivalents, refers to the compositions having affinity for each other.“Specific binding” is where the binding is selective between twomolecules. A particular example of specific binding is that which occursbetween an antibody and an antigen. Typically, specific binding can bedistinguished from non-specific when the dissociation constant (K_(D))is less than about 1×10⁻⁵ M or less than about 1×10⁻⁶M or 1×10⁻⁷ M.Specific binding can be detected, for example, by ELISA,immunoprecipitation, coprecipitation, with or without chemicalcrosslinking, two-hybrid assays and the like. Appropriate controls canbe used to distinguish between “specific” and “non-specific” binding.

In one embodiment, the antibody or antigen binding fragment binds itstarget with a Kd within the 0.1 nM range.

In one embodiment, the antibody or antigen-binding fragment thereofprovided herein comprises a modification. In another embodiment, themodification minimizes conformational changes during the shift fromdisplayed to secreted forms of the antibody or antigen-binding fragment.It is to be understood by a skilled artisan that the modification can bea modification known in the art to impart a functional property thatwould not otherwise be present if it were not for the presence of themodification. The invention encompasses antibodies which aredifferentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques, includingbut not limited, to specific chemical cleavage by cyanogen bromide,trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation,formylation, oxidation, reduction, metabolic synthesis in the presenceof tunicamycin, etc.

In another

In another embodiment, the modification is one as further defined hereinbelow. In some embodiments, the modification is a N-terminusmodification. In another embodiment, the modification is a C-terminalmodification. In yet another embodiment, the modification is anN-terminus biotinylation. In yet another embodiment, the modification isan C-terminus biotinylation. In one embodiment, the secretable form ofthe antibody or antigen-binding fragment comprises an N-terminalmodification that allows binding to an Immunoglobulin (Ig) hinge region.In another embodiment, the Ig hinge region is from but is not limitedto, an IgA hinge region. In another embodiment, the secretable form ofthe antibody or antigen-binding fragment comprises an N-terminalmodification that allows binding to an enzymatically biotinylatablesite. In another embodiment, the secretable form of the antibody orantigen-binding fragment comprises an C-terminal modification thatallows binding to an enzymatically biotinylatable site. In anotherembodiment biotinylation of said site functionilizes the site to bind toany surface coated with streptavidin, avidin, avidin-derived moieties,or a secondary reagent.

In another embodiment, the secondary reagent is a protein, a peptide, acarbohydrate, or a glycoprotein.

In one embodiment, biotinylating scFv-78 at the N-terminus generatesbiobody-78. In another embodiment, biobody-78 strongly binds to celllines transduced with human TEM1 and cells that express high andmoderate levels of endogenous human or mouse TEM1. In anotherembodiment, biotinylating said scFv-137 at the N-terminus generatesbiobody-137.

In another embodiment, an N-terminal modification of the antibody orantigen-binding fragment provided herein allows fusion of the antibodyor antigen-binding fragment with a glycoprotein on the surface of ayeast cell. In another embodiment, the glycoprotein is a proteininvolved in yeast mating. In yet another embodiment, the glycoprotein isone involved in ligand/receptor interactions. In another embodiment, theglycoprotein includes but is not limited to an Aga2. In anotherembodiment, the antibodies or antigen-binding fragments from theyeast-display library are not biotinylated. In another embodiment,antibodies or antigen-binding fragments from the yeast-display areattached to a yeast surface via a glycoprotein. In yet anotherembodiment, the glycoprotein is an Aga2 or any glycoprotein known in theart to be useful for binding said antibodies or antigen-bindingfragments to a yeast surface.

In one embodiment, an “isolated peptide” or “polypeptide” refers to anantibody or antigen-binding fragment as further provided herein. Inanother embodiment, when in reference to any polypeptide of thisinvention, the term is meant to include native polypeptides (eitherdegradation products, synthetically synthesized peptides or recombinantpeptides) and peptidomimetics (typically, synthetically synthesizedpeptides), such as peptoids and semipeptoids which are peptide analogs,which may have, for example, modifications rendering the polypeptidesmore stable while in a body or more capable of penetrating into cells.Such modifications include, but are not limited to N terminal, Cterminal or peptide bond modification, including, but not limited to,backbone modifications, and residue modification, each of whichrepresents an additional embodiment of the invention. Methods forpreparing peptidomimetic compounds are well known in the art and arespecified, for example, in Quantitative Drug Design, C A Ramsden Gd.,Chapter 17.2, F. Choplin Pergamon Press (1992). In one embodiment, apolypeptide is a full length protein or a variant of a known protein.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression.

In one embodiment, the term “polypeptide” generally refers to theantibody, antigen-binding fragments or variants of the presentinvention.

In one embodiment, the polypeptide of this invention comprises an aminoacid substitution. In one embodiment, the amino acid substitution isconservative. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). In another embodiment, the amino acid substitution is not aconservative one that results in enhanced activity of the mutatedpolypeptide compared to the native polypeptide.

The antibodies or antigen-binding fragments of this invention can beproduced by any synthetic or recombinant process such as is well knownin the art. The antibodies or antigen-binding fragments of the inventioncan further be modified to alter biophysical or biological properties bymeans of techniques known in the art. For example, the polypeptide canbe modified to increase its stability against proteases, or to modifyits lipophilicity, solubility, or binding affinity to its nativereceptor.

In some embodiments, antibody fragments may be prepared by proteolytichydrolysis of the antibody or by expression in E. coli or mammaliancells (e.g. Chinese hamster ovary cell culture or other proteinexpression systems) of DNA encoding the fragment. Antibody fragmentscan, in some embodiments, be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly. These methods are described, for example,by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety. See also Porter, R. R., Biochem. J., 73: 119-126, 1959.Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

A “variant” of a polypeptide, antibody, or protein of the presentinvention, in one embodiment, refers to an amino acid sequence that isaltered with respect to the referenced polypeptide, antibody, or proteinby one or more amino acids. In the present invention, a variant of apolypeptide retains the antibody-binding property of the referencedprotein. In another embodiment, a “variant” refers to theantigen-binding fragment of the present invention. In yet anotherembodiment, the variant is a variant of the antigen-binding fragmentthat retains specificity for a target or marker. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). In another embodiment, the variants have conservative aminoacid substitutions at one or more predicted non-essential amino acidresidues. In another embodiment, a “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a side chain with a similar charge, where, inother embodiments, the opposite is the case for “non-conservativesubstitutions”. Families of amino acid residues having side chains withsimilar charges have been defined in the art, These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Alternatively, mutations can be introduced randomly alongall or part of the coding sequence, such as by saturation mutagenesis,and the resultant mutants can be screened for biological activity toidentify mutants that retain activity. Following mutagenesis, theencoded protein may routinely be expressed and the functional and/orbiological activity of the encoded protein, can be determined usingtechniques described herein or by routinely modifying techniques knownin the art. Analogous minor variations may also include amino aciddeletions or insertions, or both. Guidance in determining which aminoacid residues may be substituted, inserted, or deleted withoutabolishing immunological reactivity may be found using computer programswell known in the art, for example, DNASTAR software.

In one embodiment, the antibody or antigen-binding fragment providedherein has a mutation in the light chain (VL). In another embodiment,the mutation is a conservative mutation. In another embodiment, themutation is a non-conservative one. In another embodiment, the mutationpresent in the VL chain is a serine to leucine point mutation inframework region (FR) 1. In another embodiment, the mutation present inthe VL chain of the antigen-binding fragment is a glycine to valinepoint mutation in framework region 3. In another embodiment, themutation present in the VL chain of the antigen-binding fragment is aleucine to methionine mutation in the complementarity determining region(CDR) 2.

In one embodiment, the term “framework region” or “FR” are thosevariable domain residues other than the hypervariable region residues.The framework regions have been precisely defined. See, e.g., Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, US Dept.Health and Human Services, National Institutes of Health, USA (5th ed.1991). Each variable domain typically has four FRs identified as FR1,FR2, FR3 and FR4. In some embodiments, “FR” also refers to an antibodyvariable region comprising amino acid residues abutting or proximal to,but outside of the CDR regions i.e. regions which directly interact withthe antigen, acting as the recognition element of the antibody moleculewithin the variable region of an antibody. In one embodiment, the term“framework region” is intended to mean each domain of the framework thatis separated by the CDRs. In some embodiments, the sequences of theframework regions of different light or heavy chains are relativelyconserved within a species. The combined heavy and light chain frameworkregions of an antibody serve to position and align the CDRs for properbinding to the antigen.

In one embodiment, the term “CDR” or “complementarity determiningregion” refers to amino acid residues comprising non-contiguous antigencombining sites found within the variable region of both heavy and lightchain polypeptides. In other embodiments, the term “CDR” will compriseregions as described by Kabat et al., J. Biol. Chem. 252, 6609-6616(1977) and Kabat et al., Sequences of protein of immunological interest.(1991), and Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) andMacCallum et al., J. Mol. Biol. 262:732-745 (1996). The amino acids ofthe CDRs of the variable domains were initially defined by Kabat, basedon sequence variability, to consist of amino acid residues 31-35B (H1),50-65 (H2), and 95-102 (H3) in the human heavy chain variable domain(VH) and amino acid residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) inthe human light chain variable domain (VL), using Kabat's numberingsystem for amino acid residues of an antibody. See Kabat et al.,sequences of proteins of immunological interest, US Dept. Health andHuman Services, NIH, USA (5th ed. 1991). Chothia and Lesk, J. Mol. Biol.196:901-917 (1987) presented another definition of the CDRs based onresidues that included in the three-dimensional structural loops of thevariable domain regions, which were found to be important in antigenbinding activity. Chothia et al. defined the CDRs as consisting of aminoacid residues 26-32 (H1), 52-56 (H2), and 95-102 (H3) in the human heavychain variable domain (VH), and amino acid residues 24-34 (L1), 50-56(L2), and 89-97 (L3) in the human light chain variable domain (VL).Combining the CDR definitions of Kabat and Chothia, the CDRs consist ofamino acid residues 26-35B (H1), 50-65 (H2), and 95-102 (H3) in human VHand amino acid residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in humanVL, based on Kabat's numbering system.

In some embodiments, a “variable region” when used in reference to anantibody or a heavy or light chain thereof is intended to mean the aminoterminal portion of an antibody which confers antigen binding onto themolecule and which is not the constant region. The term is intended toinclude functional fragments thereof which maintain some of all of thebinding function of the whole variable region. Therefore, the term“heteromeric variable region binding fragments” is intended to mean atleast one heavy chain variable region and at least one light chainvariable regions or functional fragments thereof assembled into aheteromeric complex. Heteromeric variable region binding fragmentsinclude, for example, functional fragments such as Fab, F(ab)₂, Fv,single chain Fv (scfv) and the like. Such functional fragments are wellknown to those skilled in the art. Accordingly, the use of these termsin describing functional fragments of a heteromeric variable region isintended to correspond to the definitions well known to those skilled inthe art. Such terms are described in, for example, Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York(1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference(Myers, R. A. (ed.), New York: VCH Publisher, Inc.); Huston et al., CellBiophysics, 22:189-224 (1993); Plückthun and Skerra, Meth. Enzymol.,178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry, SecondEd., Wiley-Liss, Inc., New York, N.Y. (1990).

In one embodiment the polypeptide of this invention is an isoform of theisolated polypeptide. In one embodiment, “isoform” refers to a versionof a molecule, for example, a protein or polypeptide of the presentinvention, with only slight differences to another isoform of the sameprotein or polypeptide. In one embodiment, isoforms are produced fromdifferent but related genes, or in another embodiment, arise from thesame gene by alternative splicing. In another embodiment, isoforms arecaused by single nucleotide polymorphisms.

In one embodiment the isolated polypeptide of this invention is afragment of the native protein. In one embodiment, “fragment” refers toa protein or polypeptide that is shorter or comprises fewer amino acidsthan the full length protein or polypeptide. In another embodiment,fragment refers to a nucleic acid that is shorter or comprises fewernucleotides than the full length nucleic acid. In another embodiment,the fragment is an N-terminal fragment. In another embodiment, thefragment is a C-terminal fragment. In one embodiment, the fragment ofthis invention is an intrasequential section of the protein, peptide, ornucleic acid. In another embodiment, the fragment is a functionalintrasequential section of the protein, peptide or nucleic acid. Inanother embodiment, the fragment is a functional intrasequential sectionwithin the protein, peptide or nucleic acid. In another embodiment, thefragment is an N-terminal functional fragment. In one embodiment, thefragment is a C-terminal functional fragment.

In one embodiment, the term “functional fragment” refers to a fragmentthat maintains a certain degree of biological activity as compared tothe wild type despite it being a modified version of the native or wildtype antibody or polypeptide. This degree of activity could range frommoderate to high as compared to the wild type, where the “activity”refers to its natural biophysical or biochemical characteristics, e.g.binding ability, affinity, half-life, etc.

In one embodiment, an isolated polypeptide of this invention comprises aderivate of a polypeptide of this invention. “Derivative” is to beunderstood as referring, in some embodiments, to less than thefull-length portion of the native sequence of the protein in question.In some embodiments, a “derivative” may further comprise (at its terminiand/or within said sequence itself) non-native sequences, i.e. sequenceswhich do not form part of the native protein in question. The term“derivative” also includes within its scope molecular species producedby conjugating chemical groups to the amino residue side chains of thenative proteins or fragments thereof, wherein said chemical groups donot form part of the naturally-occurring amino acid residues present insaid native proteins.

In one embodiment, the invention provides polynucleotides comprising, oralternatively consisting of, a nucleotide sequence encoding an antibodyof the invention (including molecules comprising, or alternativelyconsisting of, antibody fragments or variants thereof). The inventionalso encompasses polynucleotides that hybridize under high stringency,or alternatively, under intermediate or lower stringency hybridizationconditions, e.g., as defined supra, to polynucleotides complementary tonucleic acids having a polynucleotide sequence that encodes an antibodyof the invention or a fragment or variant thereof.

In another embodiment, the polynucleotides are obtained, and thenucleotide sequence of the polynucleotides determined, by any methodknown in the art. Alternatively, a polynucleotide encoding an antibody(including molecules comprising, or alternatively consisting of,antibody fragments or variants thereof) are generated from nucleic acidfrom a suitable source. If a clone containing a nucleic acid encoding aparticular antibody is not available, but the sequence of the antibodymolecule is known, a nucleic acid encoding the immunoglobulin may bechemically synthesized or obtained from a suitable source (e.g., anantibody cDNA library, or a cDNA library generated from, or nucleicacid, preferably poly A+RNA, isolated from, any tissue or cellsexpressing the antibody, such as hybridoma cells selected to express anantibody of the invention) by PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence toidentify, e.g. a cDNA clone from a cDNA library that encodes theantibody Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art

Methods of making antibodies and antibody fragments are known in theart. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, New York, 1988, incorporated herein byreference).

Antibodies can be produced by the immunization of various animals,including mice, rats, rabbits, goats, primates, humans and chickens witha target antigen such as TEM1 or peptide fragments of TEM1 containingthe anti-TEM1 epitope of the present invention. In one embodiment, theantibody or antigen-binding fragment is purified prior to immunizationof the animal. In one embodiment, the antibody or antigen-bindingfragment of the present invention can be purified by methods known inthe art, for example, gel filtration, ion exchange, affinitychromatography, etc. Affinity chromatography or any of a number of othertechniques known in the art can be used to isolate polyclonal ormonoclonal antibodies from serum, ascites fluid, or hybridomasupernatants.

“Purified” means that the monoclonal antibody is separated from at leastsome of the proteins normally associated with the monoclonal antibodyand preferably separated from all cellular materials other thanproteins.

In some embodiments, the term “nucleic acid” refers to polynucleotide orto oligonucleotides such as deoxyribonucleic acid (DNA), and, whereappropriate, ribonucleic acid (RNA) or mimetic thereof. The term shouldalso be understood to include, as equivalents, analogs of either RNA orDNA made from nucleotide analogs, and, as applicable to the embodimentbeing described, single (sense or antisense) and double-strandedpolynucleotides. This term includes oligonucleotides composed ofnaturally occurring nucleobases, sugars and covalent internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions, which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

As will be appreciated by one skilled in the art, a fragment orderivative of a nucleic acid sequence or gene that encodes for a proteinor peptide can still function in the same manner as the entire, wildtype gene or sequence. Likewise, forms of nucleic acid sequences canhave variations as compared to wild type sequences, neverthelessencoding a protein or peptide, or fragments thereof, retaining wild typefunction exhibiting the same biological effect, despite thesevariations. Each of these represents a separate embodiment of thepresent invention.

The nucleic acids of the present invention can be produced by anysynthetic or recombinant process such as is well known in the art.Nucleic acids according to the invention can further be modified toalter biophysical or biological properties by means of techniques knownin the art. For example, the nucleic acid can be modified to increaseits stability against nucleases (e.g., “end-capping”), or to modify itslipophilicity, solubility, or binding affinity to complementarysequences.

Methods for modifying nucleic acids to achieve specific purposes aredisclosed in the art, for example, in Sambrook et al. (1989). Moreover,the nucleic acid sequences of the invention can include one or moreportions of nucleotide sequence that are non-coding for the protein ofinterest. The invention further provides DNA sequences which encodeproteins similar to those encoded by sequences as described herein, butwhich differ in terms of their codon sequence due to the degeneracy ofthe genetic code or allelic variations (naturally-occurring base changesin the species population which may or may not result in an amino acidchange), which may encode the proteins of the invention describedherein, as well. Variations in the DNA sequences, which are caused bypoint mutations or by induced modifications (including insertion,deletion, and substitution) to enhance the activity, half-life orproduction of the polypeptides encoded thereby, are also encompassed inthe invention.

DNA encoding the antibodies or antigen-binding fragments provided hereinis readily isolated and sequenced using conventional procedures (e.g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the heavy and light chains of the antibodies).Hybridoma cells serve as a source of such DNA. Once isolated, the DNAmay be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, yeast cells or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of theantibodies in the recombinant host cells. Recombinant production ofantibodies is described in more detail below.

It is to be understood by a skilled artisan that the antibody,antigen-binding fragments, or compositions provided herein can be usedin therapeutic or diagnostic procedures.

In one embodiment, provided herein is a method of treating a tumor in asubject, whereby the method comprises the step of contacting said tumorcell with a therapeutically effective amount of an antibody orantigen-binding fragment provided herein that is operably linked to abiologically active agent.

In one embodiment, the term “operably linked” refers to thepositioning/linking of the two or more molecules or sequences in such amanner as to ensure the proper function or expression of the moleculeand sequence.

In one embodiment, the term “therapeutically effective amount” refers toan amount that provides a therapeutic effect for a given condition andadministration regimen. In the present invention, the therapeutic effectis an increase in erythrocyte levels, which can be evidenced by a risein hematocrit in the patient being treated.

In one embodiment, the term “preventing, or treating” refers to any oneor more of the following: delaying the onset of symptoms, reducing theseverity of symptoms, reducing the severity of an acute episode,reducing the number of symptoms, reducing the incidence ofdisease-related symptoms, reducing the latency of symptoms, amelioratingsymptoms, reducing secondary symptoms, reducing secondary infections,prolonging patient survival, preventing relapse to a disease, decreasingthe number or frequency of relapse episodes, increasing latency betweensymptomatic episodes, increasing time to sustained progression,expediting remission, inducing remission, augmenting remission, speedingrecovery, or increasing efficacy of or decreasing resistance toalternative therapeutics. In one embodiment, “treating” refers to boththerapeutic treatment and prophylactic or preventive measures, whereinthe object is to prevent or lessen the targeted pathologic condition ordisorder as described hereinabove.

In another embodiment, “symptoms” are manifestation of a disease orpathological condition as described hereinabove.

In another embodiment, the methods provided herein further compriseproteolytic inhibitors, pharmaceutical carriers, diluents, andadjuvants.

In another embodiment, provided herein is a method of treatingangiogenesis of a solid tumor in a subject. In another embodiment, themethod comprises the step of contacting a pericyte of the solid tumorwith the antibody or antigen-binding fragment provided herein whereinthe antibody or antigen-binding fragment is operably linked to abiologically active agent provided herein.

In another embodiment, provided herein is a method of treatingangiogenesis of a solid tumor in a subject. In another embodiment, themethod comprises the step of contacting a pericyte of the solid tumorwith a pharmaceutical composition comprising the antibody orantigen-binding fragment provided herein operably linked to abiologically active agent provided herein.

In one embodiment the compositions of this invention comprise apolypeptide, antibody, or antigen-binding fragment of this invention,alone or in some embodiments, in combination with a secondpharmaceutically active agent. In one embodiment, the term“pharmaceutically active agent” refers to any medicament which satisfiesthe indicated purpose. In some embodiments, the pharmaceutically activeagent of this invention includes, but is not limited to a decongestant,antibiotic, bronchodilator, anti-inflammatory steroid, leukotrieneantagonist or histamine receptor antagonist, and the like.

In another embodiment, provided herein is a method of delivering abiologically active agent and the antibody or antigen-binding fragmentof the present invention for the treatment of a tumor in a subject. Inanother embodiment, the method comprises the step of concomitantly butindividually administering the biologically active agent and theantibody or antigen-binding fragment. In another embodiment, the methodcomprises the step of separately administering the biologically activeagent and the antibody or antigen-binding fragment.

In one embodiment, the antibody or antigen-binding fragment providedherein are themselves “biologically active”, meaning they are able toexert the biological action or an enhanced action of their correspondingparental antibodies even after modification, in particular in binding tothe target antigen, inhibiting binding of ligands to receptors, furtherin terms of modulation, in particular inhibition of antigen-mediatedsignal transduction and prophylaxis or therapy of antigen-mediateddiseases. The term “biologically active”, when used in reference to anyof the biologically active agents described herein also refers to theagent's ability to modulate the immune response in a manner that canlead to a preventive, diagnostic, or therapeutic effect as will beunderstood by a skilled artisan. In some embodiments, agents that areused to achieve this biological activity include but are not limited toa cytokine, an enzyme, a chemokine, a radioisotope, an enzymaticallyactive toxin, a therapeutic nano particle or a chemotherapeutic agent,as will be understood by a skilled artisan.

In an alternate embodiment, the polypeptides of antibodies areconjugated or operably linked so as to function in their intendedpurpose to an enzyme in order to employ Antibody Dependent EnzymeMediated Prodrug Therapy (ADEPT). ADEPT may be used by conjugating oroperably linking the antibody or Fc fusion to a prodrug-activatingenzyme that converts a prodrug (e.g. a peptidyl chemotherapeutic agent)to an active anti-cancer drug. The enzyme component of theimmunoconjugate useful for ADEPT includes any enzyme capable of actingon a prodrug in such a way so as to convert it into its more active,cytotoxic form. Enzymes that are useful in the method of this inventioninclude but are not limited to alkaline phosphatase useful forconverting phosphate-containing prodrugs into free drugs; arylsulfataseuseful for converting sulfate-containing prodrugs into free drugs;cytosine deaminase useful for converting non-toxic 5-fluorocytosine intothe anti-cancer drug, 5-fluorouracil; proteases, such as serratiaprotease, thermolysin, subtilisin, carboxypeptidases and cathepsins(such as cathepsins B and L), that are useful for convertingpeptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases,useful for converting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuramimidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with α-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, for example, Massey, 1987,Nature 328: 457-458). Polypeptide/antibody-abzyme conjugates can beprepared for delivery of the abzyme to a tumor cell population. Otheradditional modifications of the modified molecules provided herein arecontemplated herein. For example, the polypeptide/antibody may be linkedto one of a variety of nonproteinaceous polymers, e.g., polyethyleneglycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers ofpolyethylene glycol and polypropylene glycol.

In another embodiment, the antibody/polypeptide provided herein isadministered with one or more immunomodulatory agents. Such agents mayincrease or decrease production of one or more cytokines, up- ordown-regulate self-antigen presentation, mask MHC antigens, or promotethe proliferation, differentiation, migration, or activation state ofone or more types of immune cells. Immunomodulatory agents include butare not limited to: non-steroidal anti-inflammatory drugs (NSAIDs) suchas aspirin, ibuprofen, celecoxib, diclofenac, etodolac, fenoprofen,indomethacin, ketoralac, oxaprozin, nabumentone, sulindac, tolmentin,rofecoxib, naproxen, ketoprofen, and nabumetone; steroids (e.g.glucocorticoids, dexamethasone, cortisone, hydroxycortisone,methylprednisolone, prednisone, prednisolone, trimcinolone,azulfidineicosanoids such as prostaglandins, thromboxanes, andleukotrienes; as well as topical steroids such as anthralin,calcipotriene, clobetasol, and tazarotene); cytokines such as TGFb,IFNa, IFNb, IFNg, IL-2, IL-4, IL-10; cytokine, chemokine, or receptorantagonists including antibodies, soluble receptors, and receptor-Fcfusions against BAFF, B7, CCR2, CCR5, CD2, CD3, CD4, CD6, CD7, CD8,CD11, CD14, CD15, CD17, CD18, CD20, CD23, CD28, CD40, CD40L, CD44, CD45,CD52, CD64, CD80, CD86, CD147, CD152, complement factors (C5, D) CTLA4,eotaxin, Fas, ICAM, ICOS, IFN-α IFN-β, IFN-γ, IFNAR, IgE, IL-1, IL-2,IL-2R, IL-4, IL-5R, IL-6, IL-8, IL-9 IL-12, IL-13, IL-13R1, IL-15,IL-18R, IL-23, integrins, LFA-1, LFA-3, MHC, selectins, TGF-β, TNF-α,TNF-β, TNF-R1, T-cell receptor, including Enbrel®. (etanercept),Humira®. (adalimumab), and Remicade®. (infliximab); heterologousanti-lymphocyte globulin; other immunomodulatory molecules such as2-amino-6-aryl-5 substituted pyrimidines, anti-idiotypic antibodies forMHC binding peptides and MHC fragments, azathioprine, brequinar,bromocryptine, cyclophosphamide, cyclosporine A, D-penicillamine,deoxyspergualin, FK506, glutaraldehyde, gold, hydroxychloroquine,leflunomide, malononitriloamides (e.g. leflunomide), methotrexate,minocycline, mizoribine, mycophenolate mofetil, rapamycin, andsulfasasazine.

In an alternate embodiment, antibodies of the present invention areadministered with a cytokine. By “cytokine” as used herein is meant ageneric term for proteins released by one cell population that act onanother cell as intercellular mediators. Examples of such cytokines arelymphokines, monokines, and traditional polypeptide hormones. Includedamong the cytokines are growth hormones such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor; fibroblast growth factor; prolactin; placental lactogen;tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance;mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrin; thrombopoietin (TPO); nerve growthfactors such as NGF-beta; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growthfactor-I and -II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-alpha, beta, and -gamma; colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosisfactor such as TNF-alpha or TNF-beta; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell culture,and biologically active equivalents of the native sequence cytokines.

A chemotherapeutic or other cytotoxic agent may be administered as aprodrug. The term “prodrug” refers to a precursor or derivative form ofa pharmaceutically active substance that is less cytotoxic to tumorcells compared to the parent drug and is capable of being enzymaticallyactivated or converted into the more active parent form. See, forexample Wilman, 1986, Biochemical Society Transactions, 615th MeetingBelfast, 14:375-382; and Stella et al., “Prodrugs: A Chemical Approachto Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al.,(ed.): 247-267, Humana Press, 1985. The prodrugs that may find use withthe compositions and methods as provided herein include but are notlimited to phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,beta-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use with the antibodies/polypeptides of thecompositions and methods provided herein include but are not limited toany of the aforementioned chemotherapeutic agents.

In some embodiments, any combination of the antibody/polypeptide withthe biological active agents specified above, i.e., a cytokine, anenzyme, a chemokine, a radioisotope, an enzymatically active toxin, or achemotherapeutic agent can be applied. In another embodiment, theantibody/polypeptide can be operably-linked with the biologically activeagent and used in the methods described herein or antibody/polypeptideprovided herein can merely be used in combination with the biologicallyactive agents, in a manner in which both are administered separately(i.e.—not conjugated) to achieve the desired preventive, diagnostic, ortherapeutic effect.

In one embodiment, provided herein is a method of inhibiting orsuppressing a tumor in a subject. In another embodiment, the methodcomprises the step of administering an effective amount of the antibodyor antigen-binding fragment of the present invention.

In another embodiment, provided herein is a method of delayingprogression of a solid tumor in a subject. In yet another embodiment,the method comprises administering to the subject an effective amount ofthe antibody or antigen-binding fragment thereof provided herein. Inanother embodiment, the subject mounts an immune response against apericyte of a vasculature of the solid tumor, thereby delayingprogression of the solid tumor in the subject.

In one embodiment, provided herein is a method of diagnosing thepresence of a tumor or a cancer growth in a subject. In anotherembodiment, the method comprises sampling a tissue sample isolated fromthe subject with a composition comprising the antibody orantigen-binding fragment provided herein, whereby specific binding ofsaid antibody or antigen-binding fragment to the tissue sample isindicative of the presence of a tumor or cancer growth in the subject.In another embodiment, the method further comprises detecting asecondary reagent that specifically binds to the antibody orantigen-binding fragment but does not antagonize binding of the antibodyor antigen-binding fragment to its target. In another embodiment, the“secondary reagent” is a photoactivatable agent, a fluorophore, aradioisotope, a bioluminescent protein, a bioluminescent peptide, afluorescent tag, a fluorescent protein, or a fluorescent peptide.

In one embodiment, the term “cancer” and “cancerous” refer to ordescribe, in one embodiment, the physiological condition in mammals thatis typically characterized by unregulated cell growth. Examples ofcancer include but are not limited to carcinoma, lymphoma, blastoma,sarcoma (including liposarcoma), neuroendocrine tumors, mesothelioma,schwanoma, meningioma, adenocarcinoma, melanoma, and leukemia orlymphoid malignancies.

In one embodiment, the term “cancer” includes but is not limited to,ovarian cancers, breast cancers, glioblastoma, gastrointestinal cancers.In another embodiment, the cancer is orthotopic ovarian cancer. Inanother embodiment, the ovarian cancer cell line MOV1 is used as a modelof ovarian cancer in the present invention. It is to be understood by askilled artisan that the invention is not limited to this cell line butincludes other model cells lines available in the art.

In another embodiment, “sampling” comprises the step of testing oranalyzing the sample using a detection assay that enables the detectionof a secondary reagent that is complexed with or conjugated to theantibody or antigen-binding fragment and emits a detectable “signal”when the antibody or antigen-binding fragment is specifically bound tothe target. In another embodiment, the detection is achieved usingassays routinely used in the art such as, but not limited toimmunological assays (for e.g., immunohistochemistry, ELISA, etc.) ormicroscopic imaging.

In one embodiment, the term “labeled” refers to antibodies of theinvention having one or more elements, isotopes, or chemical compoundsattached to enable the detection in a screen. In general, labels fallinto three classes: a) immune labels, which may be an epitopeincorporated as a fusion partner that is recognized by an antibody, b)isotopic labels, which may be radioactive or heavy isotopes, and c)small molecule labels, which may include fluorescent and calorimetricdyes, or molecules such as biotin that enable other labeling methods. Inone embodiment, antibodies of the invention are labeled with biotin. Inother related embodiments, biotinylated antibodies of the invention maybe used, for example, as an imaging agent or as a means of identifyingone or more ligand molecules. In another embodiment, the label can be ananoparticle that can be detected or visualized once bound to theantibody or antigen-binding fragment. Labels may be incorporated intothe compound at any position and may be incorporated in vitro or in vivoduring protein expression.

In one embodiment, the conjugate formed by the antibody orantigen-binding fragment and the secondary reagent provided herein areused for various applications such as, but not limited to, flowcytometry, ELISA, Western blotting, immunohistochemistry, membraneassays, and diagnostic and therapeutic methods as further describedherein or as routinely applied in the art.

In one embodiment, an antibody of the present invention is administeredto a patient having a disease involving inappropriate expression of atarget antigen, a protein or other molecule. Within the scope of thepresent invention this is meant to include diseases and disorderscharacterized by aberrant proteins, due for example to alterations inthe amount of a protein present, protein localization, posttranslationalmodification, conformational state, the presence of a mutant or pathogenprotein, etc. Similarly, the disease or disorder may be characterized byalterations molecules including but not limited to polysaccharides andgangliosides. An overabundance may be due to any cause, including butnot limited to overexpression at the molecular level, prolonged oraccumulated appearance at the site of action, or increased activity of aprotein relative to normal. Included within this definition are diseasesand disorders characterized by a reduction of a protein. This reductionmay be due to any cause, including but not limited to reduced expressionat the molecular level, shortened or reduced appearance at the site ofaction, mutant forms of a protein, or decreased activity of a proteinrelative to normal. Such an overabundance or reduction of a protein canbe measured relative to normal expression, appearance, or activity of aprotein, and said measurement may play an important role in thedevelopment and/or clinical testing of the antibodies of the presentinvention.

In one embodiment, provided herein is a method of imaging aTEM1-containing tumor. In another embodiment, the method comprises thestep of applying the antibody or antigen-binding fragment providedherein that is operably linked to a secondary reagent. In anotherembodiment, the secondary reagent can be visualized once the antibody orantigen-binding fragment has bound its target. In yet anotherembodiment, the secondary reagent is a photoactivatable agent, afluorophore, a radioisotope, a bioluminescent protein, a bioluminescentpeptide, a fluorescent tag, a fluorescent protein, or a fluorescentpeptide. Non-limiting examples of secondary reagents are provided below.

In one embodiment, the detectable label or secondary reagent attachedthereto, include labels such as, but not limited to a fluorescent label(e.g., fluorescein, isothiocyanate (FITC), a cyanine dye, etc.), anaffinity label (e.g., biotin, avidin, protein A, etc.), an enzymaticlabel (e.g., horseradish peroxidase or alkaline phosphatase), or anisotopic label (e.g., ¹²⁵I) or any other such detectable moiety to allowfor detection and isolation of the antibody.

Detection methods for identification of binding species within thepopulation of altered variable regions can be direct or indirect and caninclude, for example, the measurement of light emission, radioisotopes,calorimetric dyes and fluorochromes. Direct detection includes methodsthat operate without intermediates or secondary measuring procedures toassess the amount of bound antigen or ligand. Such methods generallyemploy ligands that are themselves labeled by, for example, radioactive,light emitting or fluorescent moieties. In contrast, indirect detectionincludes methods that operate through an intermediate or secondarymeasuring procedure. These methods generally employ molecules thatspecifically react with the antigen or ligand and can themselves bedirectly labeled or detected by a secondary reagent. For example, aantibody specific for a ligand can be detected using a secondaryantibody capable of interacting with the first antibody specific for theligand, again using the detection methods described above for directdetection. Indirect methods can additionally employ detection byenzymatic labels. Moreover, for the specific example of screening forcatalytic antibodies, the disappearance of a substrate or the appearanceof a product can be used as an indirect measure of binding affinity orcatalytic activity.

In specific embodiments, antibodies of the invention are labeled withEuropium. For example, antibodies of the invention may be labelled withEuropium using the DELFIA Eu-labeling kit (catalog #1244-302, PerkinElmer Life Sciences, Boston, Mass.) following manufacturer'sinstructions.

In specific embodiments, antibodies of the invention are attached tomacrocyclic chelators useful for conjugating radiometal ions, includingbut not limited to, 111In, 177Lu, 90Y, 166Ho, 153Sm, 215Bi and 225Ac topolypeptides. In a preferred embodiment, the radiometal ion associatedwith the macrocyclic chelators attached to antibodies of the inventionis 111In. In another preferred embodiment, the radiometal ion associatedwith the macrocyclic chelator attached to antibodies polypeptides of theinvention is 90Y. In specific embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA). Inspecific embodiments, the macrocyclic chelator is.quadrature.-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraaza-cyclodo-decane-1,4,7,10-tetraaceticacid. In other specific embodiments, the DOTA is attached to theantibody of the invention via a linker molecule. Examples of linkermolecules useful for conjugating a macrocyclic chelator such as DOTA toa polypeptide are commonly known in the art—see, for example, DeNardo etal., Clin Cancer Res. 4(10):2483-90, 1998; Peterson et al., Bioconjug.Chem. 10(4):553-7, 1999; and Zimmerman et al, Nucl. Med. Biol.26(8):943-50, 1999 which are hereby incorporated by reference in theirentirety. In addition, U.S. Pat. Nos. 5,652,361 and 5,756,065, whichdisclose chelating agents that may be conjugated to antibodies, andmethods for making and using them, are hereby incorporated by referencein their entireties.

Also provided by the invention are chemically modified derivatives ofantibodies of the invention which may provide additional advantages suchas increased solubility, stability and in vivo or in vitro circulatingtime of the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The antibodies may be modified at randompositions within the molecule, or at predetermined positions within themolecule and may include one, two, three or more attached chemicalmoieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2560, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the antibody with consideration of effects on functional orantigenic domains of the antibody. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include, for example, lysineresidues and the N-terminal amino acid residues; those having a freecarboxyl group may include aspartic acid residues, glutamic acidresidues, and the C-terminal amino acid residue. Sulfhydryl groups mayalso be used as a reactive group for attaching the polyethylene glycolmolecules. Preferred for therapeutic purposes is attachment at an aminogroup, such as attachment at the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins,e.g., antibodies, via linkage to any of a number of amino acid residues.For example, polyethylene glycol can be linked to a proteins viacovalent bonds to lysine, histidine, aspartic acid, glutamic acid, orcysteine residues. One or more reaction chemistries may be employed toattach polyethylene glycol to specific amino acid residues (e.g.,lysine, histidine, aspartic acid, glutamic acid, or cysteine) of theprotein or to more than one type of amino acid residue (e.g., lysine,histidine, aspartic acid, glutamic acid, cysteine and combinationsthereof) of the protein.

In one embodiment, provided herein is a method of biotinylating theantibodies or antigen-binding fragments provided herein. In anotherembodiment, the method comprises the step of mating theantibody-secreting yeast with a biotin ligase-bearing yeast. It is to beunderstood by a skilled artisan that other modifications some of whichare provided herein and others which are routinely carried out in theart are encompassed within the invention.

In another embodiment, provided herein is a method of optimizingtransformation and construct expression efficiency of a yeast strain. Inanother embodiment the method comprises the step of resuspending theyeast at room temperature, washing the yeast with water and lithiumacetate at 4° C., incubating the yeast with the transforming vectorwithout dimethyl sulfoxide (DMSO), heat shocking the yeast at 42° C. for30 min, and allowing the yeast to recover in Yeast Peptone Dextrose(YPD) media for 2 hr.

In one embodiment, provided herein is a method of optimizingtransformation and construct expression efficiency of a yeast strain. Inanother embodiment, the method comprises the step of preparing theconstruct under in the presence of 25 mM dithiothreitol (DTT), 20 mMHepes, 0.6M sorbitol, followed by shaking for 30 min at 30° C.; and,transforming the yeast strain with the construct.

In one embodiment provided herein is a method of screening for andidentifying an antibody or antigen-binding fragment thereof withoptimized affinity for a tumor target/marker. In another embodiment, themethod comprises displaying the antibody or antigen-binding fragmentthereof on the surface of a yeast cell, testing its binding affinity toa marker or a pool of makers, and identifying the antibody orantigen-binding fragment that binds with the highest affinity to adesired marker.

In another embodiment, the “marker” is human or murine TEM1. In anotherembodiment, the antibody or antigen-binding fragment selectively bindswith a high affinity to its target. In another embodiment, the range ofthe high affinity binding is between 0.05 to 0.1, nM. In anotherembodiment, the range of high affinity is between 0.1 to 0.5, nM. Inanother embodiment, the range of high affinity is between 0.5 to 1.0,nM.

In another embodiment, for the purposes of the invention, the terms“tumor marker”, “tumor target”, “target”, “antigen” are all synonymousand refer to the compound/molecule that is specifically and selectivelyrecognized by the antibody or antigen-binding fragment of the presentinvention.

In one embodiment, provided herein is a method of screening for anantibody or antigen-binding fragment thereof with optimized affinity fora tumor marker. In another embodiment, the method comprises transforminga yeast with a nucleic acid sequence encoding the antibody orantigen-binding fragment in secretable form, testing the bindingaffinity of the secreted antibody or antigen-binding fragment thereof toa marker or pool of markers, and identifying an antibody orantigen-binding fragment that binds with the highest affinity to themarker. Methods for screening for an antibodies that specifically bindto a target are provided for herein below.

In another embodiment, the term “transformed” refers to a genetic changein a cell following incorporation of nucleic acid (e.g., a transgene)exogenous to the cell. Thus, a “transformed cell” is a cell into which,or a progeny of which a nucleic acid molecule has been introduced bymeans of recombinant DNA techniques. Cell transformation to produce hostcells may be carried out as described herein or using techniques knownin the art. Accordingly, methods of producing cells containing thenucleic acids and cells expressing the antibodies or antigen-bindingfragments of the invention are also provided.

In some embodiments, the present invention provides a library ofantibodies or antigen-binding fragments for use in the methods andcompositions of the present invention. Previously reported yeastlibraries have been severely limited in size, with a maximum of 5×10⁶,but typically less than 1×10⁵, transformants per microgram of DNA forcommonly used strains, resulting in insufficient diversity and potentialfor yielding high affinity antibodies. In one embodiment, providedherein is a display library of affinity-optimized antibodies orantigen-binding fragments of the present invention. In anotherembodiment, the library comprises up to 1×10⁸ transformants permicrogram of DNA. In another embodiment, the library is a yeast displaylibrary of affinity-optimized antibodies or antigen-binding fragmentsthereof.

In some embodiments, the library provided herein is a nucleic acidlibrary, a phage display library, a yeast display library or anoligopeptide library. In some embodiments, the process yields a Fabfragment library, a FR library, a VH library, a VL library, a VH and VLlibrary, a CDR library, or an ScFv yeast display library. According tothis aspect of the invention, and in some embodiments, the inventionprovides a library of affinity-optimized antibodies or antigen-bindingfragments thereof of known specificity prepared according to a processof the invention.

In another embodiment the library is a yeast library comprising asecretable form of an antibody or antigen-binding fragment of thepresent invention. In another embodiment, the library is derived from athrombotic thrombocytopenic purpura (TTP) patient.

In one embodiment, the term “library” refers to a set antibodies orantigen-binding fragments, as described herein, in any form, includingbut not limited to a list of nucleic acid or amino acid sequences, alist of nucleic acid or amino acid substitutions at variable positions,a physical library comprising nucleic acids that encode the librarysequences, or a physical library comprising the antibodies orantigen-binding fragments, either in purified or unpurified form. Inanother embodiment, the term refers to a set of antibodies orantigen-binding fragments displayed in any form as indicated above, buton the surface of a yeast cell. Accordingly, there are a variety oftechniques that may be used to efficiently generate libraries of thepresent invention. Such methods that may find use in the presentinvention are described or referenced in U.S. Pat. No. 6,403,312; U.S.Ser. Nos. 09/782,004; 09/927,790; 10/218,102; PCT WO 01/40091; and PCTWO 02/25588, all incorporated entirely by reference. Such methodsinclude but are not limited to gene assembly methods, PCR-based methodsand methods which use variations of PCR, ligase chain reaction-basedmethods, pooled oligo methods such as those used in synthetic shuffling,error-prone amplification methods and methods which use oligos withrandom mutations, classical site-directed mutagenesis methods, cassettemutagenesis, and other amplification and gene synthesis methods. As isknown in the art, there are a variety of commercially available kits andmethods for gene assembly, mutagenesis, vector subcloning, and the like,and such commercial products find use in the present invention forgenerating nucleic acids that encode the antibodies or antigen-bindingfragments display libraries provided herein.

Recombinant antibody libraries can be expressed on the surface of yeastcells, phages or bacterial cells. Methods for preparing and screeninglibraries expressed on the surface of yeast cells are described furtherin International Application Publication No. WO 99/36569. Methods forpreparing and screening libraries expressed on the surface of bacterialcells are described further in U.S. Pat. No. 6,699,658.

In one embodiment, a “yeast display library” refers to a collection ofyeast (for e.g., Saccharomyces cerevisiae, Saccharomyces pombe) whereinthe yeast express an external (typically heterologous) protein, (e.g. anscFv). The external protein is free to interact with (bind to) othermoieties with which the yeast cells are contacted. Each yeast displayingan external protein is a “member” of the yeast display library.

In some embodiments, yeast-display offers several advantages overprokaryotic systems, including superior sampling of the immune antibodyrepertoire; post-translational modifications (glycosylation) due to theeukaryotic expression; fastar and more controlled flow cytrometry-basedselection compared to solid phase panning; and absence of growth bias,as recombinant proteins are displayed at the yeast cell surface onlyduring the induction step in the presence of galactose.

In one embodiment, a “phage display library” refers to a collection ofphage (e.g., filamentous phage) wherein the phage expresses an external(typically heterologous) protein. The external protein is free tointeract with (bind to) other moieties with which the phage arecontacted. Each phage displaying an external protein is a “member” ofthe phage display library.

The term “filamentous phage” or “filamentous bacteriophage” refers to aviral particle capable of displaying a heterogenous polypeptide on itssurface. Although one skilled in the art will appreciate that a varietyof bacteriophage may be employed in the present invention, in preferredembodiments the vector is, or is derived from, a filamentousbacteriophage, such as, for example, f1, fd, Pf1, M13, etc. Thefilamentous phage may contain a selectable marker such as tetracycline(e.g., “fd-tet”). Various filamentous phage display systems are wellknown to those of skill in the art (see, e.g., Zacher et al. (1980) Gene9: 127-140, Smith et al.(1985) Science 228: 1315-1317 (1985); andParmley and Smith (1988) Gene 73: 305-318).

An assembly cell is a cell in which a nucleic acid can be packaged intoa viral coat protein (capsid). Assembly cells may be infected with oneor more different virus particles (e.g. a normal or debilitated phageand a helper phage) that individually or in combination direct packagingof a nucleic acid into a viral capsid.

In one embodiment, phage display is used to create the ScFv variantlibrary. In another embodiment, the method of preparing a phage displaylibrary comprises in some embodiments, the steps of modifying a phagemidvector for cloning, assembling VH and VL variable FR regions, followedby sequencing analysis, sequential cloning of VL and VH into thephagemid vector, and building a large size library. In anotherembodiment, the library of VH and VL gene segments from yeast displayscFv libraries are constructed consist of both the Framework resides(FR1-FR4) interspersed with CDRs 1-3.

Introduction of nucleic acid encoding an antibody or antigen-bindingfragment into target cells can also be carried out by conventionalmethods known in the art such as osmotic shock (e.g., calciumphosphate), electroporation, microinjection, cell fusion, etc.Introduction of nucleic acid and polypeptide in vitro, ex vivo and invivo can also be accomplished using other techniques. For example, apolymeric substance, such as polyesters, polyamine acids, hydrogel,polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers, polylactide/glycolide copolymers, or ethylenevinylacetatecopolymers. A nucleic acid can be entrapped in microcapsules prepared bycoacervation techniques or by interfacial polymerization, for example,by the use of hydroxymethylcellulose or gelatin-microcapsules, or poly(methylmethacrolate) microcapsules, respectively, or in a colloid drugdelivery system. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, beads, and lipid-based systems,including oil-in-water emulsions, micelles, mixed micelles, andliposomes.

The use of liposomes for introducing various compositions into cells,including nucleic acids, is known to those skilled in the art (see,e.g., U.S. Pat. Nos. 4,844,904, 5,000,959, 4,863,740, and 4,975,282). Acarrier comprising a natural polymer, or a derivative or a hydrolysateof a natural polymer, described in WO 94/20078 and U.S. Pat. No.6,096,291, is suitable for mucosal delivery of molecules, such aspolypeptides and polynucleotides. Piperazine based amphilic cationiclipids useful for gene therapy also are known (see, e.g., U.S. Pat. No.5,861,397). Cationic lipid systems also are known (see, e.g., U.S. Pat.No. 5,459,127). Accordingly, viral and non-viral vector means ofdelivery into cells or tissue, in vitro, in vivo and ex vivo areincluded.

In one embodiment, nucleotide sequences can be “operably linked”, i.e.,positioned, to ensure the functioning of an expression control sequence.These expression constructs are typically replicable in the cells eitheras episomes or as integral parts of the cell's chromosomal DNA, and maycontain appropriate origins of replication for the respectiveprokaryotic strain employed for expression. Commonly, expressionconstructs contain selection markers, such as for example, tetracyclineresistance, ampicillin resistance, kanamycin resistance orchlormaphenicol resistance, facilitating detection and/or selection ofthose bacterial cells transformed with the desired nucleic acidsequences (see, e.g., U.S. Pat. No. 4,704,362). These markers, however,are not exclusionary, and numerous others may be employed, as known tothose skilled in the art. In another embodiment of the present inventionexpression constructs contain both positive and negative selectionmarkers.

Similarly reporter genes may be incorporated within expressionconstructs to facilitate identification of transcribed products.Accordingly, in one embodiment of the present invention, reporter genesutilized are selected from the group consisting of β-galactosidase,chloramphenicol acetyl transferase, luciferase and a fluorescentprotein.

Prokaryotic promoter sequences regulate expression of the encodedpolynucleotide sequences, and in some embodiments of the presentinvention, are operably linked to polynucleotides encoding thepolypeptides of this invention. In additional embodiments of the presentinvention, these promoters are either constitutive or inducible, andprovide a means of high and low levels of expression of the polypeptidesof this invention, and in some embodiments, for regulated expression ofmultiple polypeptides of the invention, which in some embodiments areexpressed as a fusion protein.

Many well-known bacterial promoters, including the T7 promoter system,the lactose promoter system, typtophan (Trp) promoter system, Trc/TacPromoter Systems, beta-lactamase promoter system, tetA Promoter systems,arabinose regulated promoter system, Phage T5 Promoter, or a promotersystem from phage lambda, may be employed, and others, as well, andcomprise embodiments of the present invention. The promoters willtypically control expression, optionally with an operator sequence andmay include ribosome binding site sequences for example, for initiatingand completing transcription and translation. According to additionalembodiments, the vector may also contain expression control sequences,enhancers that may regulate the transcriptional activity of thepromoter, appropriate restriction sites to facilitate cloning of insertsadjacent to the promoter and other necessary information processingsites, such as RNA splice sites, polyadenylation sites and transcriptiontermination sequences as well as any other sequence which may facilitatethe expression of the inserted nucleic acid.

In another embodiment, the present invention comprises methods of use ofa polynucleotide, vector, polypeptide and/or fragment thereof as hereindescribed and/or compositions comprising the same in treating,inhibiting or preventing.

In another embodiment, the present invention comprises methods of use ofa polynucleotide, vector, antibodies and/or fragment thereof as hereindescribed and/or compositions comprising the same in treating,inhibiting or preventing.

Sequence alignment methods that can be used to achieve the desiredsequence alignment include in some embodiments, but are not solelyrestricted to pair-wise alignment methods or multiple-sequence alignmentmethods, as will be understood by a skilled artisan. Sequence alignmentscan be stored in a wide variety of text-based file formats. In oneembodiment, this is achieved by converting in certain embodiments, anyformat, for example a FASTA or GenBank, SwissProt, Entrez and EMBLformat, using conversion programs and programming packages such as,READSEQ, EMBOSS and BioPerl, BioRuby. It is to be understood that askilled artisan can convert, modify, score, update and/or store thesequences as necessary using any program or storage media, as will beappreciated by the skilled artisan.

In some embodiments, the term “sequence alignment” includes use of anyprogram or method, as understood by a skilled artisan, that is used toperform nucleic acid or amino acid sequence alignments to yield resultsthat can be readily probed, assessed and subjected to mathematical andstatistical calculations. In one embodiment, methods for sequence orstructure alignment are well known in the art, and include alignmentsbased on sequence and structural homology, as will be understood by askilled artisan.

In one embodiment, the term “homology,” “homolog” or “homologous” referto sequence identity, or to structural identity, or functional identity.By using the term “homology” and the other like forms, it is to beunderstood that any molecule, whether nucleic acid or peptide, thatfunctions similarly, and/or contains sequence identity, and/or isconserved structurally so that it approximates the reference sequence,is to be considered as part of this invention. In another embodiment,the terms “homology”, “homologue” or “homologous”, in any instance,indicate that the sequence referred to, whether an amino acid sequence,or a nucleic acid sequence, exhibits at least 86% correspondence withthe indicated sequence. In another embodiment, the amino acid sequenceor nucleic acid sequence exhibits at least 90% correspondence with theindicated sequence. In another embodiment, the amino acid sequence ornucleic acid sequence exhibits at least 92% correspondence with theindicated sequence. In another embodiment, the amino acid sequence ornucleic acid sequence exhibits at least 95% correspondence with theindicated sequence. In another embodiment, the amino acid sequence ornucleic acid sequence exhibits at least 95% or more correspondence withthe indicated sequence. In another embodiment, the amino acid sequenceor nucleic acid sequence exhibits at least 97% or more correspondencewith the indicated sequence. In another embodiment, the amino acidsequence or nucleic acid sequence exhibits 97%-100% correspondence tothe indicated sequence. In another embodiment, the amino acid sequenceor nucleic acid sequence exhibits 100% correspondence to the indicatedsequence. Similarly, in one embodiment, the reference to acorrespondence to a particular sequence includes both directcorrespondence, as well as homology to that sequence as herein defined.Accordingly and in one embodiment, the term “non-homologous” refers theamino acid sequence or nucleic acid sequence exhibits no more than 85%correspondence with the indicated sequence. In another embodiment, theamino acid sequence or nucleic acid sequence exhibits no more than 75%correspondence with the indicated sequence. In another embodiment, theamino acid sequence or nucleic acid sequence exhibits no more than65-74% correspondence with the indicated sequence. In anotherembodiment, the amino acid sequence or nucleic acid sequence exhibits nomore than 55-64% correspondence with the indicated sequence. In anotherembodiment, the amino acid sequence or nucleic acid sequence exhibits nomore than 45-54% correspondence with the indicated sequence. In anotherembodiment, the amino acid sequence or nucleic acid sequence exhibits nomore than 35-44% correspondence with the indicated sequence. In anotherembodiment, the amino acid sequence or nucleic acid sequence exhibits nomore than 35-44% correspondence with the indicated sequence. In anotherembodiment, the amino acid sequence or nucleic acid sequence exhibits nomore than 15-34% correspondence with the indicated sequence. In anotherembodiment, the amino acid sequence or nucleic acid sequence exhibits nomore than 5-14% correspondence with the indicated sequence. In anotherembodiment, the amino acid sequence or nucleic acid sequence exhibits nomore than 0.1-4% correspondence with the indicated sequence. In anotherembodiment, the term “non-homologous can be used interchangeably withthe term “low sequence similarity”.

In one embodiment, the invention also provides transformed cells andprogeny thereof into which a nucleic acid molecule encoding an antibodyor antigen-binding fragment, has been introduced by means of recombinantDNA techniques in vitro, ex vivo or in vivo. The transformed cells canbe propagated and the introduced nucleic acid transcribed, or encodedprotein expressed. It is understood that a progeny cell may not beidentical to the parental cell, since there may be mutations that occurduring replication. Transformed cells include but are not limited toprokaryotic and eukaryotic cells such as bacteria, fungi, plant, insect,and animal (e.g., mammalian, including human) cells. The cells may bepresent in culture, in a cell, tissue or organ ex vivo or present in asubject.

Typically cell transformation employs a vector. The term “vector,”refers to, e.g., a plasmid, virus, such as a viral vector, or othervehicle known in the art that can be manipulated by insertion orincorporation of a nucleic acid, for genetic manipulation (i.e.,“cloning vectors”), or can be used to transcribe or translate theinserted polynucleotide (i.e., “expression vectors”). Such vectors areuseful for introducing nucleic acids, including a nucleic acid thatencodes a humanized antibody operably linked with an expression controlelement, and expressing the encoded protein in vitro (e.g., in solutionor in solid phase), in cells or in vivo.

In one embodiment, the expression vector(s) is(are) transferred to ahost cell by conventional techniques and the transfected cells are thencultured by conventional techniques to produce an antibody orantigen-binding fragment of the invention. Thus, the invention includeshost cells containing polynucleotide(s) encoding an antibody of theinvention (e.g., whole antibody, a heavy or light chain thereof, orportion thereof, or a single chain antibody, or a fragment or variantthereof), operably linked to a heterologous promoter. In otherembodiments, for the expression of entire antibody molecules, vectorsencoding both the heavy and light chains are co-expressed in the hostcell for expression of the entire immunoglobulin molecule.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude, but are not limited to, bacteriophage particles engineered toexpress antibody fragments or variants thereof (single chainantibodies), microorganisms such as bacteria (e.g., E. coli, B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing antibody coding sequences;yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293, 3T3, NS0 cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably,bacterial cells such as Escherichia coli, and more preferably,eukaryotic cells, especially for the expression of whole recombinantantibody molecule, are used for the expression of a recombinant antibodymolecule. For example, mammalian cells such as Chinese hamster ovarycells (CHO), in conjunction with a vector such as the major intermediateearly gene promoter element from human cytomegalovirus is an effectiveexpression system for antibodies (Foecking et al., Gene 45:101 (1986);Cockett et al., Bio/Technology 8:2 (11990); Bebbington et al.,Bio/Techniques 10:169 (1992); Keen and Hale, Cytotechnology 18:207(1996)). These references are incorporated in their entireties byreference herein.

A vector used to transform a cell or a host-expression vector generallycontains at least an origin of replication for propagation in the cell.Control elements, including expression control elements as set forthherein, present within a vector, are included to facilitatetranscription and translation. The term “expression control element” isintended to include, at a minimum, one or more components whose presencecan influence expression, and can include components other than or inaddition to promoters or enhancers, for example, leader sequences andfusion partner sequences, internal ribosome binding sites (IRES)elements for the creation of multigene, or polycistronic, messages,splicing signal for introns, maintenance of the correct reading frame ofthe gene to permit in-frame translation of mRNA, polyadenylation signalto provide proper polyadenylation of the transcript of a gene ofinterest, stop codons, etc.

Vectors can include a selection marker. As is known in the art,“selection marker” means a gene that allows for the selection of cellscontaining the gene. “Positive selection” refers to a process wherebyonly cells that contain the selection marker will survive upon exposureto the positive selection. Drug resistance is one example of a positiveselection marker; cells containing the marker will survive in culturemedium containing the selection drug, and cells which do not contain themarker will die. Such markers include drug resistance genes such as neo,which confers resistance to G418, hygr, which confers resistance tohygromycin, or puro which confers resistance to puromycin, among others.Other positive selection marker genes include genes that allowidentification or screening of cells containing the marker. These genesinclude genes for fluorescent proteins (GFP), the lacZ gene, thealkaline phosphatase gene, and surface markers such as CD8, amongothers.

Vectors can contain negative selection markers. “Negative selection”refers to a process whereby cells containing a negative selection markerare killed upon exposure to an appropriate negative selection agent. Forexample, cells which contain the herpes simplex virus-thymidine kinase(HSV-tk) gene (Wigler et al., Cell 11:223 (1977)) are sensitive to thedrug gancyclovir (GANC). Similarly, the gpt gene renders cells sensitiveto 6-thioxanthine.

Mammalian expression systems further include vectors specificallydesigned for in vivo and ex vivo expression. Such systems includeadeno-associated virus (AAV) vectors (U.S. Pat. No. 5,604,090). AAVvectors have previously been shown to provide expression of Factor IX inhumans and in mice at levels sufficient for therapeutic benefit (Kay etal., Nat. Genet. 24:257 (2000); Nakai et al., Blood 91:4600 (1998)).Adenoviral vectors (U.S. Pat. Nos. 5,700,470, 5,731,172 and 5,928,944),herpes simplex virus vectors (U.S. Pat. No. 5,501,979) and retroviral(e.g., lentivirus vectors are useful for infecting dividing as well asnon-dividing cells and foamy virues) vectors (U.S. Pat. Nos. 5,624,820,5,693,508, 5,665,577, 6,013,516 and 5,674,703 and WIPO publicationsWO92/05266 and WO92/14829) and papilloma virus vectors (e.g., human andbovine papilloma virus) have all been employed in gene therapy (U.S.Pat. No. 5,719,054). Vectors also include cytomegalovirus (CMV) basedvectors (U.S. Pat. No. 5,561,063). Vectors that efficiently delivergenes to cells of the intestinal tract have been developed and also maybe used (see, e.g., U.S. Pat. Nos. 5,821,235, 5,786,340 and 6,110,456).In yeast, vectors that facilitate integration of foreign nucleic acidsequences into a chromosome, via homologous recombination, for example,are known in the art and can be used. Yeast artificial chromosomes (YAC)are typically used when the inserted nucleic acids are too large formore conventional vectors (e.g., greater than about 12 kb).

In one embodiment, phagemid vectors for use in the invention include anyavailable in the art suitable for the production of theantibodies/antibody templates/FR libraries of the present invention andinclude phagemid vectors pCB04, pIT1, pIT2, CANTAB 6, pComb 3 HS.Filamentous vectors and methods of phagemid construction are describedin, for example, U.S. Pat. Nos. 6,054,312 and 6,803,230, eachincorporated herein by reference. Bacteriophage display systemsinvolving non-filamentous bacteriophage vectors known as cytoplasmicbacteriophage or lytic phage can also be utilized as described in forexample, U.S. Pat. No. 5,766,905, incorporated herein by reference.

Suitable bacterial expression constructs for use with the presentinvention include, but are not limited to the pCAL, pUC, pET, pETBlue™(Novagen), pBAD, pLEX, pTrcHis2, pSE280, pSE380, pSE420 (Invitrogen),pKK223-2 (Clontech), pTrc99A, pKK223-3, pRIT2T, pMC1871, pEZZ 18(Pharmacia), pBluescript II SK (Stratagene), pALTER-Ex1, pALTER-Ex2,pGEMEX (Promega), pFivE (MBI), pQE (Qiagen) commercially availableexpression constructs, and their derivatives, and others known in theart. In some embodiments of the present invention the construct may alsoinclude, a virus, a plasmid, a bacmid, a phagemid, a cosmid, or abacteriophage.

In one embodiment, provided herein is a vector encoding the displaylibrary of the present invention. In another embodiment, the vector ispAGA2.

In another embodiment, provided herein is a vector encoding thesecretable library of the present invention. In another embodiment, thevector is p416BCCP.

Antibodies may be screened using a variety of methods, including but notlimited to those that use in vitro assays, in vivo and cell-basedassays, and selection technologies. Properties of antibodies that may bescreened include but are not limited to stability, solubility, andaffinity for the target. Multiple properties may be screenedsimultaneously or individually. Proteins may be purified or unpurified,depending on the requirements of the assay. In one embodiment, thescreen is a qualitative or quantitative binding assay for binding ofantibodies to a protein or nonprotein molecule that is known or thoughtto bind the antibody. In one embodiment, the screen is a binding assayfor measuring binding to the target antigen. Automation andhigh-throughput screening technologies may be utilized in the screeningprocedures. Screening may employ the use of a fusion partner or label.Binding assays can be carried out using a variety of methods known inthe art, including but not limited to FRET (Fluorescence ResonanceEnergy Transfer) and BRET (Bioluminescence Resonance EnergyTransfer)-based assays, AlphaScreen.™. (Amplified Luminescent ProximityHomogeneous Assay), Scintillation Proximity Assay, ELISA (Enzyme-LinkedImmunosorbent Assay), SPR (Surface Plasmon Resonance, also known asBiacore.™), isothermal titration calorimetry, differential scanningcalorimetry, gel electrophoresis, and chromatography including gelfiltration. These and other methods may take advantage of some fusionpartner or label of the antibody. Assays may employ a variety ofdetection methods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels.

In some embodiments, the screening of populations of polypeptides suchas the altered variable region populations produced by the methods ofthe invention, involve immobilization of the populations of alteredvariable regions to filters or other solid substrate. This isparticularly advantageous because large numbers of different species canbe efficiently screened for antigen binding. Such filter lifts willallow for the identification of altered variable regions that exhibitsubstantially the same or greater binding affinity compared to the donorvariable region. Alternatively, if the populations of altered variableregions are expressed on the surface of a cell, a yeast orbacteriophage, for example, panning on immobilized antigen can be usedto efficiently screen for the relative binding affinity of specieswithin the population.

Another affinity method for screening populations of altered variableregions polypeptides is a capture lift assay that is useful foridentifying a binding molecule having selective affinity for a ligand(Watkins et. al., (1997)). This method employs the selectiveimmobilization of altered variable regions to a solid support and thenscreening of the selectively immobilized altered variable regions forselective binding interactions against the cognate antigen or bindingpartner. Selective immobilization functions to increase the sensitivityof the binding interaction being measured since initial immobilizationof a population of altered variable regions onto a solid support reducesnon-specific binding interactions with irrelevant molecules orcontaminants which can be present in the reaction.

Another method for screening populations or for measuring the affinityof individual altered variable region polypeptides is through surfaceplasmon resonance (SPR). This method is based on the phenomenon whichoccurs when surface plasmon waves are excited at a metal/liquidinterface. Light is directed at, and reflected from, the side of thesurface not in contact with sample, and SPR causes a reduction in thereflected light intensity at a specific combination of angle andwavelength. Biomolecular binding events cause changes in the refractiveindex at the surface layer, which are detected as changes in the SPRsignal. The binding event can be either binding association ordisassociation between a receptor-ligand pair. The changes in refractiveindex can be measured essentially instantaneously and therefore allowsfor determination of the individual components of an affinity constant.More specifically, the method enables accurate measurements ofassociation rates (kon) and disassociation rates (koff). Methods formeasuring the affinity, including association and disassociation ratesusing surface plasmon resonance are well known in the arts and can befound described in, for example, Jonsson and Malmquist, Advances inBiosnsors, 2:291-336 (1992) and Wu et al. Proc. Natl. Acad. Sci. USA,95:6037-6042 (1998). Moreover, one apparatus well known in the art formeasuring binding interactions is a BIAcore 2000 instrument which iscommercially available through Pharmacia Biosensor, (Uppsala, Sweden).

The pharmacokinetics (PK) of the antibodies of the invention can bestudied in a variety of animal systems, with the most relevant beingnon-human primates such as the cynomolgus, rhesus monkeys. Single orrepeated i.v./s.c. administrations over a dose range of 6000-fold(0.05-300 mg/kg) can be evaluated for the half-life (days to weeks)using plasma concentration and clearance as well as volume ofdistribution at a steady state and level of systemic absorbance can bemeasured. Examples of such parameters of measurement generally includemaximum observed plasma concentration (Cmax), the time to reach Cmax(Tmax), the area under the plasma concentration-time curve from time 0to infinity [AUC(0-inf] and apparent elimination half-life (T_(1/2)).Additional measured parameters could include compartmental analysis ofconcentration-time data obtained following i.v. administration andbioavailability.

In another embodiment, toxicity studies are performed to determine theantibody effects that cannot be evaluated in standard pharmacologyprofile or occur only after repeated administration of the agent. Mosttoxicity tests are performed in two species—a rodent and a non-rodent—toensure that any unexpected adverse effects are not overlooked before newtherapeutic entities are introduced into man. In general, these modelsmay measure a variety of toxicities including genotoxicity, chronictoxicity, immunogenicity, reproductive/developmental toxicity andcarcinogenicity. Included within the aforementioned parameters arestandard measurement of food consumption, bodyweight, antibodyformation, clinical chemistry, and macro- and microscopic examination ofstandard organs/tissues (e.g. cardiotoxicity). Additional parameters ofmeasurement are injection site trauma and the measurement ofneutralizing antibodies, if any. Traditionally, monoclonal antibodytherapeutics, naked or conjugated are evaluated for cross-reactivitywith normal tissues, immunogenicity/antibody production, conjugate orlinker toxicity and “bystander” toxicity of radiolabeled species.Nonetheless, such studies may have to be individualized to addressspecific concerns and following the guidance set by ICH S6 (Safetystudies for biotechnological products also noted above). As such, thegeneral principles are that the products are sufficiently wellcharacterized and for which impurities/contaminants have been removed,that the test material is comparable throughout development, and GLPcompliance.

The biophysical properties of antibodies, for example stability andsolubility, may be screened using a variety of methods known in the art.Protein stability may be determined by measuring the thermodynamicequilibrium between folded and unfolded states. For example, antibodiesof the present invention may be unfolded using chemical denaturant,heat, or pH, and this transition may be monitored using methodsincluding but not limited to circular dichroism spectroscopy,fluorescence spectroscopy, absorbance spectroscopy, NMR spectroscopy,calorimetry, and proteolysis. As will be appreciated by those skilled inthe art, the kinetic parameters of the folding and unfolding transitionsmay also be monitored using these and other techniques. The solubilityand overall structural integrity of an antibody may be quantitatively orqualitatively determined using a wide range of methods that are known inthe art. Methods which may find use in the present invention forcharacterizing the biophysical properties of antibodies include gelelectrophoresis, isoelectric focusing, capillary electrophoresis,chromatography such as size exclusion chromatography, ion-exchangechromatography, and reversed-phase high performance liquidchromatography, peptide mapping, oligosaccharide mapping, massspectrometry, ultraviolet absorbance spectroscopy, fluorescencespectroscopy, circular dichroism spectroscopy, isothermal titrationcalorimetry, differential scanning calorimetry, analyticalultra-centrifugation, dynamic light scattering, proteolysis, andcross-linking, turbidity measurement, filter retardation assays,immunological assays, fluorescent dye binding assays, protein-stainingassays, microscopy, and detection of aggregates via ELISA or otherbinding assay. Structural analysis employing X-ray crystallographictechniques and NMR spectroscopy may also find use. In one embodiment,stability and/or solubility is measured by determining the amount ofprotein solution after some defined period of time. In this assay, theprotein may or may not be exposed to some extreme condition, for exampleelevated temperature, low pH, or the presence of denaturant. Becausefunction typically requires a stable, soluble, and/orwell-folded/structured protein, the aforementioned functional andbinding assays also provide ways to perform such a measurement. Forexample, a solution comprising an antibody could be assayed for itsability to bind target antigen, then exposed to elevated temperature forone or more defined periods of time, then assayed for antigen bindingagain. Because unfolded and aggregated protein is not expected to becapable of binding antigen, the amount of activity remaining provides ameasure of the antibody's stability and solubility.

The biological properties of the antibodies of the present invention maybe further characterized in cell, tissue, and whole organismexperiments. As is known in the art, drugs are often tested in animals,including but not limited to mice, rats, rabbits, dogs, cats, pigs, andmonkeys, in order to measure a drug's efficacy for treatment against adisease or disease model, or to measure a drug's pharmacokinetics,toxicity, and other properties. Said animals may be referred to asdisease models. With respect to the antibodies of the present invention,a particular challenge arises when using animal models to evaluate thepotential for in-human efficacy of candidate polypeptides—this is due,at least in part, to the fact that antibodies that have a specificeffect on the affinity for a human Fc receptor may not have a similaraffinity effect with the orthologous animal receptor. These problems canbe further exacerbated by the inevitable ambiguities associated withcorrect assignment of true orthologs (Mechetina et al., Immunogenetics,2002 54:463-468, incorporated entirely by reference), and the fact thatsome orthologs simply do not exist in the animal (e.g. humans possess anFcγRIIa whereas mice do not). Therapeutics are often tested in mice,including but not limited to nude mice, SCID mice, xenograft mice, andtransgenic mice (including knockins and knockouts). For example, anantibody of the present invention that is intended as an anti-cancertherapeutic may be tested in a mouse cancer model, for example axenograft mouse. In this method, a tumor or tumor cell line is graftedonto or injected into a mouse, and subsequently the mouse is treatedwith the therapeutic to determine the ability of the antibody to reduceor inhibit cancer growth and metastasis. Such experimentation mayprovide meaningful data for determination of the potential of saidantibody to be used as a therapeutic. Any organism, e.g., mammals, maybe used for testing. For example because of their genetic similarity tohumans, monkeys can be suitable therapeutic models, and thus may be usedto test the efficacy, toxicity, pharmacokinetics, or other property ofthe antibodies of the present invention. Tests of the antibodies of thepresent invention in humans are ultimately required for approval asdrugs, and thus of course these experiments are contemplated. Thus theantibodies of the present invention may be tested in humans to determinetheir therapeutic efficacy, toxicity, pharmacokinetics, and/or otherclinical properties.

In one embodiment, the library provided herein is screened using one ormore cell-based or in vitro assays. For such assays, antibodies,purified or unpurified, are typically added exogenously such that cellsare exposed to individual variants or groups of variants belonging to alibrary. These assays are typically, but not always, based on thebiology of the ability of the antibody to bind to antigen and mediatesome biochemical event, for example effector functions like cellularlysis, phagocytosis, ligand/receptor binding inhibition, inhibition ofgrowth and/or proliferation, apoptosis, etc. Such assays often involvemonitoring the response of cells to antibody, for example cell survival,cell death, cellular phagocytosis, cell lysis, change in cellularmorphology, or transcriptional activation such as cellular expression ofa natural gene or reporter gene. For example, such assays may measurethe ability of antibodies to elicit ADCC, ADCP, or CDC. For some assaysadditional cells or components, that is in addition to the target cells,may need to be added, for example serum complement, or effector cellssuch as peripheral blood monocytes (PBMCs), NK cells, macrophages, andthe like. Such additional cells may be from any organism, e.g., humans,mice, rats, rabbits, monkeys, etc. Crosslinked or monomeric antibodiesmay cause apoptosis of certain cell lines expressing the antibody'starget antigen, or they may mediate attack on target cells by immunecells which have been added to the assay. Methods for monitoring celldeath or viability are known in the art, and include the use of dyes,fluorophores, immunochemical, cytochemical, and radioactive reagents.For example, caspase assays or annexin-flourconjugates may enableapoptosis to be measured, and uptake or release of radioactivesubstrates (e.g. Chromium-51 release assays) or the metabolic reductionof fluorescent dyes such as alamar blue may enable cell growth,proliferation, or activation to be monitored. In one embodiment, theDELFIA® EuTDA-based cytotoxicity assay (Perkin Elmer, Mass.) is used.Alternatively, dead or damaged target cells may be monitored bymeasuring the release of one or more natural intracellular proteins, forexample lactate dehydrogenase. Transcriptional activation may also serveas a method for assaying function in cell-based assays. In this case,response may be monitored by assaying for natural genes or proteinswhich may be upregulated or down-regulated, for example the release ofcertain interleukins may be measured, or alternatively readout may bevia a luciferase or GFP-reporter construct. Cell-based assays may alsoinvolve the measure of morphological changes of cells as a response tothe presence of an antibody. Cell types for such assays may beprokaryotic or eukaryotic, and a variety of cell lines that are known inthe art may be employed. Alternatively, cell-based screens are performedusing cells that have been transformed or transfected with nucleic acidsencoding the antibodies. In one embodiment, example of such assays areprovided herein, (see FIG. 8 and Example 5).

A variety of selection methods are known in the art that may find use inthe present invention for screening protein libraries. These include butare not limited to yeast display, yeast-two-hybrid based screening. Inother embodiments, further methods include phage display (Phage displayof peptides and proteins: a laboratory manual, Kay et al., 1996,Academic Press, San Diego, Calif., 1996; Lowman et al., 1991,Biochemistry 30:10832-10838; Smith, 1985, Science 228:1315-1317) and itsderivatives such as selective phage infection (Malmborg et al., 1997, JMol Biol 273:544-551), selectively infective phage (Krebber et al.,1997, J Mol Biol 268:619-630), and delayed infectivity panning (Benharet al., 2000, J Mol Biol 301:893-904), cell surface display (Witrrup,2001, Curr Opin Biotechnol, 12:395-399) such as display on bacteria(Georgiou et al., 1997, Nat Biotechnol 15:29-34; Georgiou et al., 1993,Trends Biotechnol 11:6-10; Lee et al., 2000, Nat Biotechnol 18:645-648;June et al., 1998, Nat Biotechnol 16:576-80), yeast (Boder & Wittrup,2000, Methods Enzymol 328:430-44; Boder & Wittrup, 1997, Nat Biotechnol15:553-557), and mammalian cells (Whitehorn et al., 1995, Bio/technology13:1215-1219), as well as in vitro display technologies (Amstutz et al.,2001, Curr Opin Biotechnol 12:400-405) such as polysome display(Mattheakis et al., 1994, Proc Natl Acad Sci USA 91:9022-9026), ribosomedisplay (Hanes et al., 1997, Proc Natl Acad Sci USA 94:4937-4942), mRNAdisplay (Roberts & Szostak, 1997, Proc Natl Acad Sci USA 94:12297-12302;Nemoto et al., 1997, FEBS Lett 414:405-408), and ribosome-inactivationdisplay system (Zhou et al., 2002, J Am Chem Soc 124, 538-543).

Other selection methods that may find use in the present inventioninclude methods that do not rely on display, such as in vivo methodsincluding but not limited to periplasmic expression and cytometricscreening (Chen et al., 2001, Nat Biotechnol 19:537-542), the proteinfragment complementation assay (Johnsson & Varshaysky, 1994, Proc NatlAcad Sci USA 91:10340-10344; Pelletier et al., 1998, Proc Natl Acad SciUSA 95:12141-12146), and the yeast two hybrid screen (Fields & Song,1989, Nature 340:245-246) used in selection mode (Visintin et al, 1999,Proc Natl Acad Sci USA 96:11723-11728). In an alternate embodiment,selection is enabled by a fusion partner that binds to a specificsequence on the expression vector, thus linking covalently ornoncovalently the fusion partner and associated variant library memberwith the nucleic acid that encodes them. For example, U.S. Ser. Nos.09/642,574; 10/080,376; 09/792,630; 10/023,208; 09/792,626; 10/082,671;09/953,351; 10/097,100; 60/366,658; PCT WO 00/22906; PCT WO 01/49058;PCT WO 02/04852; PCT WO 02/04853; PCT WO 02/08023; PCT WO 01/28702; andPCT WO 02/07466 describe such a fusion partner and technique that mayfind use in the present invention. In an alternative embodiment, in vivoselection can occur if expression of the protein imparts some growth,reproduction, or survival advantage to the cell.

Methods of identifying antibodies through their binding affinities orspecificities are very well known in the art and include methods such asimmunoprecipitation or an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Other well-known methods can be used to determine antibody bindingaffinities and these methods can be readily used, as will be understoodby a skilled artisan. In that regard, the method of the presentinvention further comprises determining a respective binding affinityfor a target for each of said antibodies in said library formed. Inanother embodiment the method further comprises identifying an antibodyhaving the highest binding affinity for said target. According to thisaspect, and in one embodiment, this invention provides a humanizedantibody optimized for affinity to a known target identified by themethods of this invention. Antibodies with known specificity areprepared and their affinity assessed. Antibodies with known specificitywhose affinity is desired to be optimized by the methods of thisinvention may be constructed by any means known in the art. For example,monoclonal antibodies may be produced in a number of ways, includingusing the hybridoma method (e.g. as described by Kohler et al., Nature,256: 495, 1975, herein incorporated by reference), or by recombinant DNAmethods (e.g., U.S. Pat. No. 4,816,567, herein incorporated byreference).

Once an antibody of interest has been identified from a library, DNAsencoding the light and heavy chains of the antibody can be isolated bystandard molecular biology techniques, such as by polymerase chainreaction (PCR) amplification of DNA from the display package (e.g.,phage, yeast) isolated during the library screening process. Nucleotidesequences of antibody light and heavy chain genes from whicholigonucleotide primers can be prepared are known in the art. Forexample, many such sequences are disclosed in Kabat et al. (1991) supraand in the “Vbase” human germline sequence database, administered by theMRC Centre for Protein Engineering (Cambridge, UK).

The antibodies of the present invention may find use in a wide range ofproducts. In one embodiment the antibody of the invention is atherapeutic, a diagnostic, or a research reagent. In one embodiment, anantibody of the invention is a therapeutic. In some embodiments, theantibody or antigen-binding fragment of the present invention is usedfor agricultural or industrial uses. An antibody of the presentinvention may find use in an antibody composition that is monoclonal orpolyclonal. The antibodies of the present invention may be agonists,antagonists, neutralizing, inhibitory, or stimulatory. In oneembodiment, the antibodies of the present invention are used to killtarget cells that bear the target antigen, for example cancer cells. Inan alternate embodiment, the antibodies of the present invention areused to block, antagonize, or agonize the target antigen. In analternate embodiment, the antibodies of the present invention are usedto block, antagonize, or agonize the target antigen and kill the targetcells that bear the target antigen. In another embodiment, the targetcell is a tumor cell or it's pericyte. In one embodiment, pericytes areplay an important role in angiogenesis and are also considered to be atarget of the antibodies or antigen-binding fragments provided herein.

The invention also provides a kit for preparing a library of humanantibody templates. In this regard, the kit comprises a library ofpolynucleotides encoding human antibody templates comprising FrameworkRegion (FR) regions possessing residues comprising a positivecomplementarity determining region (CDR) contact ratio score and apositive human diversity score or a positive B to M score and a positivehuman diversity score as described herein above.

The invention provides a kit for preparing a library of antibody orantigen-binding fragments thereof. In one embodiment, the kit comprisesa vector comprising the polynucleotides encoding the antibody orantigen-binding fragments thereof of the invention, or in anotherembodiment, the kit comprises bacteriophages comprising thepolynucleotides encoding the antibody or antigen-binding fragmentsthereof of the invention. In another embodiment, the kit comprises yeastexpressing the antibody or antigen-binding fragments thereof of theinvention

The invention further provides kits comprising one or more compositionsof the invention, including pharmaceutical formulations, packaged intosuitable packaging material. In another embodiment, a kit includes anucleic acid encoding the antibody or antigen-binding fragments thereofof the invention. In additional embodiments, a kit includes nucleicacids that further include an expression control element; an expressionvector; a viral expression vector; an adeno-associated virus expressionvector; an adenoviral expression vector; and a retroviral expressionvector. In yet an additional embodiment, a kit includes a cell that theantibody or antigen-binding fragments thereof of the invention.

In additional embodiments, a kit includes a label or packaging insertincluding instructions for expressing a humanized antibody or a nucleicacid encoding the antibody or antigen-binding fragments thereof in cellsin vitro, in vivo, or ex vivo. In yet additional embodiments, a kitincludes a label or packaging insert including instructions for treatinga subject (e.g., a subject having or at risk of having asthma) with theantibody or antigen-binding fragments thereof of the invention in vivo,or ex vivo.

As used herein, the term “packaging material” refers to a physicalstructure housing the components of the kit. The packaging material canmaintain the components sterilely, and can be made of material commonlyused for such purposes (e.g., paper, corrugated fiber, glass, plastic,foil, ampules, etc.). The label or packaging insert can includeappropriate written instructions, for example, practicing a method ofthe invention, e.g., treating the common cold. Kits of the inventiontherefore can additionally include instructions for using the kitcomponents in a method of the invention.

Instructions can include instructions for practicing any of the methodsof the invention described herein. Thus, invention pharmaceuticalcompositions can be included in a container, pack, or dispenser togetherwith instructions for administration to a subject. Instructions mayadditionally include indications of a satisfactory clinical endpoint orany adverse symptoms that may occur, or additional information requiredby the Food and Drug Administration for use on a human subject.

In one embodiment, polypeptides of the present invention areadministered as part of a vaccine. In some embodiments, the term vaccineis to be understood to encompass any immunomodulating composition, andsuch vaccines may comprise an adjuvant, an antigen, an immuno-modulatorycompound, or a combination thereof, in addition to the polypeptides ofthis invention.

In some embodiments, the adjuvant may include, but is not limited to:(A) aluminium compounds (e.g. aluminium hydroxide, aluminium phosphate,aluminium hydroxyphosphate, oxyhydroxide, orthophosphate, sulphate, etc.[e.g. see chapters 8 & 9 of ref. 96]), or mixtures of differentaluminium compounds, with the compounds taking any suitable form (e.g.gel, crystalline, amorphous, etc.), and with adsorption being preferred;(B) MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated intosubmicron particles using a microfluidizer); (C) liposomes; (D) ISCOMs,which may be devoid of additional detergent; (E) SAF, containing 10%Squalane, 0.4% Tween 80, 5% pluronic-block polymer L121, and thr-MDP,either micro fluidized into a submicron emulsion or vortexed to generatea larger particle size emulsion; (F) Ribi™ adjuvant system (RAS), (RibiImmunochem) containing 2% Squalene, 0.2% Tween 80, and one or morebacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (Detox™); (G) saponin adjuvants, suchas QuilA or QS21, also known as Stimulon™; (H) chitosan; (I) completeFreund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (J)cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophage colonystimulating factor, tumor necrosis factor, etc.; (K) monophosphoryllipid A (MPL) or 3-O-deacylated MPL (3dMPL)]; (L) combinations of 3dMPLwith, for example, QS21 and/or oil-in-water emulsions; (M)oligonucleotides comprising CpG motifs] i.e. containing at least one CGdinucleotide, with 5-methylcytosine optionally being used in place ofcytosine; (N) a polyoxyethylene ether or a polyoxyethylene ester; (O) apolyoxyethylene sorbitan ester surfactant in combination with anoctoxynol or a polyoxyethylene alkyl ether or ester surfactant incombination with at least one additional non-ionic surfactant such as anoctoxynol; (P) an immuno-stimulatory oligonucleotide (e.g. a CpGoligonucleotide) and a saponin; (Q) an immuno-stimulant and a particleof metal salt; (R) a saponin and an oil-in-water emulsion; (S) a saponin(e.g. QS21)+3dMPL+IL12 (optionally+a sterol); (T) E. coli heat-labileenterotoxin (“LT”), or detoxified mutants thereof, such as the K63 orR72 mutants; (U) cholera toxin (“CT”), or diphtheria toxin (“DT”) ordetoxified mutants of either; (V) double-stranded RNA; (W)monophosphoryl lipid A mimics, such as aminoalkyl glucosaminidephosphate derivatives e.g. RC-529]; (X) polyphosphazene (PCPP); or (Y) abioadhesive such as esterified hyaluronic acid microspheres or amucoadhesive such as crosslinked derivatives of poly(acrylic acid),polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides andcarboxymethylcellulose.

In some embodiments, administration of the compounds of this inventionis intended to reduce the severity of the pathologic condition. By theterm “reduce the severity of the pathologic condition”, it is to beunderstood that any reduction via the methods, compounds andcompositions disclosed herein, is to be considered encompassed by theinvention. Reduction in severity may, in one embodiment compriseenhancement of survival, or in another embodiment, halting diseaseprogression, or in another embodiment, delay in disease progression.

In one embodiment, dosing is dependent on the cellular responsiveness tothe administered molecules/compounds or compositions comprising same. Ingeneral, the doses utilized for the above described purposes will vary,but will be in an effective amount to exert the desired effect, asdetermined by a clinician of skill in the art. As used herein, the term“pharmaceutically effective amount” refers to an amount of a compound asdescribed herein, which will produce the desired alleviation in symptomsor other desired phenotype in a patient.

In one embodiment of the invention, the concentrations of the compoundswill depend on various factors, including the nature of the condition tobe treated, the condition of the patient, the route of administrationand the individual tolerability of the compositions.

In some embodiments, any of the compositions of this invention willcomprise a compound, in any form or embodiment as described herein. Insome embodiments, any of the compositions of this invention will consistof a compound, in any form or embodiment as described herein. In someembodiments, any of the compositions of this invention will consistessentially of a compound, in any form or embodiment as describedherein. In some embodiments, the term “comprise” refers to the inclusionof the indicated active agent, such as the compound of this invention,as well as inclusion of other active agents, and pharmaceuticallyacceptable carriers, excipients, emollients, stabilizers, etc., as areknown in the pharmaceutical industry.

In some embodiments, the compositions of this invention will consistessentially of a polypeptide/polynucleotide/vector as herein described.In some embodiments, the term “consisting essentially of” refers to acomposition whose only active ingredient of a particular class ofagents, is the indicated active ingredient, however, other compounds maybe included which are involved directly in the therapeutic effect of theindicated active ingredient. In some embodiments, the term “consistingessentially of” refers to a composition whose only active ingredient oftargeting a particular mechanism, or acting via a particular pathway, isthe indicated active ingredient, however, other compounds may beincluded which are involved directly in the therapeutic effect of theindicated active ingredient, which for example have a mechanism ofaction related to but not directly to that of the indicated agent. Insome embodiments, the term “consisting essentially of” refers to acomposition whose only active ingredient is the indicated activeingredient, however, other compounds may be included which are forstabilizing, preserving, etc. the formulation, but are not involveddirectly in the therapeutic effect of the indicated active ingredient.In some embodiments, the term “consisting essentially of” may refer tocomponents which facilitate the release of the active ingredient. Insome embodiments, the term “consisting” refers to a composition, whichcontains the active ingredient and a pharmaceutically acceptable carrieror excipient.

It will be appreciated that the actual amounts of active compound in aspecific case will vary according to the specific compound beingutilized, the particular compositions formulated, the mode ofapplication, and the particular conditions and organism being treated.Dosages for a given host can be determined using conventionalconsiderations, e.g., by customary comparison of the differentialactivities of the subject compounds and of a known agent, e.g., by meansof an appropriate, conventional pharmacological protocol.

In one embodiment, the compounds of the invention are administeredacutely for acute treatment of temporary conditions, or may beadministered chronically, especially in the case of progressive,recurrent, or degenerative disease. In one embodiment, one or morecompounds of the invention may be administered simultaneously, or inanother embodiment, they may be administered in a staggered fashion. Inone embodiment, the staggered fashion may be dictated by the stage orphase of the disease.

Parenteral vehicles (for subcutaneous, intravenous, intraarterial, orintramuscular injection) include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's and fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Examples are sterile liquids such as water and oils, with orwithout the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. In general, water, saline, aqueous dextrose andrelated sugar solutions, and glycols such as propylene glycols orpolyethylene glycol are preferred liquid carriers, particularly forinjectable solutions. Examples of oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,mineral oil, olive oil, sunflower oil, and fish-liver oil.

In one embodiment, the route of administration may be parenteral, or acombination thereof. In another embodiment, the route may beintra-ocular, conjunctival, topical, transdermal, intradermal,subcutaneous, intraperitoneal, intravenous, intra-arterial, vaginal,rectal, intratumoral, parcanceral, transmucosal, intramuscular,intravascular, intraventricular, intracranial, inhalation (aerosol),nasal aspiration (spray), intranasal (drops), sublingual, oral, aerosolor suppository or a combination thereof. In one embodiment, the dosageregimen will be determined by skilled clinicians, based on factors suchas exact nature of the condition being treated, the severity of thecondition, the age and general physical condition of the patient, bodyweight, and response of the individual patient.

For intranasal administration or application by inhalation, solutions orsuspensions of the compounds mixed and aerosolized or nebulized in thepresence of the appropriate carrier suitable. Such an aerosol maycomprise any agent described herein.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants, including suppositories and enemas.Ampoules are convenient unit dosages. Such a suppository may compriseany agent described herein.

Sustained or directed release compositions can be formulated, e.g.,liposomes or those wherein the active compound is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc. Such compositions may be formulated forimmediate or slow release. It is also possible to freeze-dry the newcompounds and use the lyophilisates obtained, for example, for thepreparation of products for injection.

For liquid formulations, pharmaceutically acceptable carriers may beaqueous or non-aqueous solutions, suspensions, emulsions or oils.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Examples of oils arethose of petroleum, animal, vegetable, or synthetic origin, for example,peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, andfish-liver oil.

In one embodiment, a composition of or used in the methods of thisinvention may be administered alone or within a composition. In anotherembodiment, compositions of this invention admixture with conventionalexcipients, i.e., pharmaceutically acceptable organic or inorganiccarrier substances suitable for parenteral, enteral (e.g., oral) ortopical application which do not deleteriously react with the activecompounds may be used. In one embodiment, suitable pharmaceuticallyacceptable carriers include but are not limited to water, saltsolutions, alcohols, gum arabic, vegetable oils, benzyl alcohols,polyethylene glycols, gelatine, carbohydrates such as lactose, amyloseor starch, magnesium stearate, talc, silicic acid, viscous paraffin,white paraffin, glycerol, alginates, hyaluronic acid, collagen, perfumeoil, fatty acid monoglycerides and diglycerides, pentaerythritol fattyacid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. Inanother embodiment, the pharmaceutical preparations can be sterilizedand if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, coloring, flavoring and/oraromatic substances and the like which do not deleteriously react withthe active compounds. In another embodiment, they can also be combinedwhere desired with other active agents, e.g., vitamins.

Pharmaceutical compositions include “pharmaceutically acceptable” and“physiologically acceptable” carriers, diluents or excipients. In oneembodiment, the terms “pharmaceutically acceptable” and “physiologicallyacceptable” refers to any formulation which is safe, and provides theappropriate delivery for the desired route of administration of aneffective amount of at least one compound for use in the presentinvention. This term refers to the use of buffered formulations as well,wherein the pH is maintained at a particular desired value, ranging frompH 4.0 to pH 9.0, in accordance with the stability of the compounds androute of administration. The terms include solvents (aqueous ornon-aqueous), solutions, emulsions, dispersion media, coatings, isotonicand absorption promoting or delaying agents, compatible withpharmaceutical administration. Such formulations can be contained in aliquid; emulsion, suspension, syrup or elixir, or solid form; tablet(coated or uncoated), capsule (hard or soft), powder, granule, crystal,or microbead. Supplementary active compounds (e.g., preservatives,antibacterial, antiviral and antifungal agents) can also be incorporatedinto the compositions.

Pharmaceutical compositions of the present invention can include one ormore further chemotherapeutic agents selected from the group consistingof nitrogen mustards (e.g., cyclophosphamide and ifosfamide), aziridines(e.g., thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g.,carmustine and streptozocin), platinum complexes (e.g., carboplatin andcisplatin), non-classical alkylating agents (e.g., dacarbazine andtemozolamide), folate analogs (e.g., methotrexate), purine analogs(e.g., fludarabine and mercaptopurine), adenosine analogs (e.g.,cladribine and pentostatin), pyrimidine analogs (e.g., fluorouracil(alone or in combination with leucovorin) and gemcitabine), substitutedureas (e.g., hydroxyurea), antitumor antibiotics (e.g., bleomycin anddoxorubicin), epipodophyllotoxins (e.g., etoposide and teniposide),microtubule agents (e.g., docetaxel and paclitaxel), camptothecinanalogs (e.g., irinotecan and topotecan), enzymes (e.g., asparaginase),cytokines (e.g., interleukin-2 and interferon-α), monoclonal antibodies(e.g., trastuzumab and bevacizumab), recombinant toxins and immunotoxins(e.g., recombinant cholera toxin-B and TP-38), cancer gene therapies,physical therapies (e.g., hyperthermia, radiation therapy, and surgery)and cancer vaccines (e.g., vaccine against telomerase).

The compositions (e.g., antibodies, and bispecific molecules) of theinvention can also be administered together with complement.Accordingly, within the scope of the invention are compositionscomprising human antibodies, multispecific or bispecific molecules andserum or complement. These compositions are advantageous in that thecomplement is located in close proximity to the human antibodies,multispecific or bispecific molecules. Alternatively, the humanantibodies, multispecific or bispecific molecules of the invention andthe complement or serum can be administered separately.

Pharmaceutical compositions can be formulated to be compatible with aparticular local or systemic route of administration. Thus,pharmaceutical compositions include carriers, diluents, or excipientssuitable for administration by particular routes. Specific non-limitingexamples of routes of administration for compositions of the inventionare inhalation or intranasal delivery. Additional routes includeparenteral, e.g., intravenous, intradermal, subcutaneous, oral,transdermal (topical), transmucosal, and rectal administration.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include: a sterile diluent such as waterfor injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide.

Pharmaceutical compositions for injection include sterile aqueoussolutions (where water soluble) or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof.Fluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Antibacterial andantifungal agents include, for example, parabens, chlorobutanol, phenol,ascorbic acid and thimerosal. Isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, sodium chloride can be includedin the composition. Including an agent which delays absorption, forexample, aluminum monostearate and gelatin can prolong absorption ofinjectable compositions.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of above ingredients followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle containing a basic dispersion medium and otheringredients as above. In the case of sterile powders for the preparationof sterile injectable solutions, methods of preparation include, forexample, vacuum drying and freeze-drying which yields a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays, inhalation devices (e.g., aspirators) orsuppositories. For transdermal administration, the active compounds areformulated into ointments, salves, gels, or creams as generally known inthe art.

The present invention's antibodies, including subsequences and modifiedforms and nucleic acids encoding them, can be prepared with carriersthat protect against rapid elimination from the body, such as acontrolled release formulation or a time delay material such as glycerylmonostearate or glyceryl stearate. The compositions can also bedelivered using implants and microencapsulated delivery systems toachieve local or systemic sustained delivery or controlled release.

Biodegradable, biocompatable polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to cells or tissues using antibodies or viral coat proteins)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

Additional pharmaceutical formulations appropriate for the compositionsfor administration in the methods of the invention are known in the art(see, e.g., Remington's Pharmaceutical Sciences (1990) 18^(th) ed., MackPublishing Co., Easton, Pa.; The Merck Index (1996) 12^(th) ed., MerckPublishing Group, Whitehouse, N.J.; and Pharmaceutical Principles ofSolid Dosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa.,(1993)). The pharmaceutical formulations can be packaged in dosage unitform for ease of administration and uniformity of dosage. “Dosage unitform” as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the pharmaceuticalcarrier or excipient.

Although the pharmaceutical compositions provided herein are principallydirected to pharmaceutical compositions which are suitable foradministration to humans, it will be understood by the skilled artisanthat such compositions are generally suitable for administration toanimals of all sorts. Modification of pharmaceutical compositionsuitable for administration to humans in order to render thecompositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with little, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, and other mammals.

It is to be understood that any amino acid sequence, whether obtainednaturally or synthetically by any means, exhibiting sequence, structuralor functional homology to the polypeptides described herein, areconsidered part of this invention.

In one embodiment, the term “about” means in quantitative terms plus orminus 5%, or in another embodiment plus or minus 10%, or in anotherembodiment plus or minus 15%, or in another embodiment plus or minus20%.

The term “subject” refers in one embodiment to a mammal including ahuman in need of therapy for, or susceptible to, a condition or itssequelae. The subject may include dogs, cats, pigs, cows, sheep, goats,horses, rats, and mice and humans. The term “subject” does not excludean individual that is normal in all respects.

It is to be understood that reference to any publication, patentapplication or issued patent is to be considered as fully incorporatedherein by reference in its entirety.

It is to be understood that any assay for measuring a particularactivity which is modulated by the therapeutic compound may be employed,as a means of determining the efficacy of the compound, in oneembodiment, optimal loading of the compound, in another embodiment,timing and dosage, in another embodiment, or a combination thereof.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention.

EXAMPLES

Materials and Methods:

Development of Companion Vectors for Yeast-Display and Yeast-Secretionof N-Terminal Biotinylated scFv

The pAGA2 vector for yeast-display (FIG. 1c ) was derived from shuttlevector p414 GAL1 (24) (the kind gift of Martin Funk, IMT, Philipps-Univ.Marburg, Germany) The pAGA2 multiple cloning site (MCS) was engineeredas follows: the first site Nhe1 was inserted after a FLAG tag and a(G₄S)₃ linker sequence. The second site EcoR1 was part of a stop codonthat is removed when cDNA is inserted in frame in the cloning site. Thethird site Xho1 was inserted just 5′ to the c-myc tag, out of frame withthe FLAG tag, to insure that both tags could be expressed only in thepresence of correctly inserted cDNA.

The pAGA2 full insert sequence is:

(SEQ ID NO: 1) 5′-agtgatgcagttacttcgctgtttttcaatattttctgttattgcttcagttttagcacaggaactgacaactatatgcgagcaaatcccctcaccaactttagaatcgacgccgtactctttgtcaacgactactattttggccaacgggaaggcaatgcaaggagtttttgaatattacaaatcagtaacgtttgtcagtaattgcggttctcacccctcaacaactagcaaaggcagccccataaacacacagtatgatttgattataaagatgacgataaaggtggtggaggtggttctggtggtggaggttctggtggtggtggatctgctagctgaattcctcgagggatccgaacaaaagcttatttctgaagaagacttgtaa-3′.The PCR product was generated from oligonucleotidetemplates and elongated by primers includingrecombination sequences for gap repair cloning: 5 ′Display(SEQ ID NO: 2) 5′-caaggagaaaaaactatatctagaactagtgatgcagttacttcgctgatttc-3′ and 3′Display (SEQ ID NO: 3)5′-gtaagcgtgacataactaattacatgactcgattacaagtcttcttcagaaataagcttttgttc-3′.

To construct the p416 BCCP vector, the companion vector of pAGA2 for thesecretion of N-terminal biotinylated scFv (FIG. 1d ), the shuttle vectorp416 GAL1 (24) (the kind gift of Martin Funk) was linearized by BamH1and Xho1, and co-transformed in yeast with a PCR product encoding alphapreproleader and RK endopeptidase sequences fused to a biotin acceptingsite (BCCP), IgA hinge, FLAG tag, (G₄S)₃ linker, cloning site with astop codon, and V5-HIS tags.

The p416 BCCP full insert sequence is:

(SEQ ID NO: 4) 5′-atgagataccacaatattactgcagattattcgcagcatcctccgcattagctgctccagtcaacactacaacaggagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgagctgattgccattaccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctttggataaaagatgtgatggtttgaatgatatttttgaagctcaaaaaattgaatggcatgaaccatcaacaccaccaactccaagtccactactcctcctacaccacaccatcagattataaagatgacgataaaggtggtggaggtggttctggtggtggaggttctggtggtggtggatctgaattcgctagctaagtcgacggtaagcctatccctaaccctctcctcggtctcgattctacgcatcatcaccatca ccat-3′.Generation of rhTEM1-GST and rGST Recombinant Proteins and ofTEM1-Transduced Cell Lines.

hTEM1 cDNA (NM_020404) was a kind gift from Dr. Ballmer-Hofer (PaulScherrer Institut, Switzerland). The 1113 bp fragment corresponding tonucleotides 75-1187 of NM_020404 was cloned into the BamH1 site of theGlutathione S-transferase Gene Fusion Vector pGEX-2TK (Life Science,Piscataway, N.J.) to obtain hTEM1-pGEX-2TK plasmid. hTEM1-pGEX-2TK andthe control vector pGEX-2TK were transformed into E. coliBL21-CodonPlus(DE3)-RIPL (Stratagene, Cedar Creek, Tex.) to producehuman TEM1 recombinant protein fused to GST (rhTEM1-GST) and GSTrecombinant protein (rGST). Transformants were inoculated into fresh 2YTmedium and incubated at 37° C. on an orbital shaker (200 rpm) overnight.Each sample was then inoculated into 500 mL of fresh medium at adilution of 1:50, and incubated in a shaking incubator at 37° C. untilthe OD600 was 0.8. Isopropyl β-d-1-thiogalactopyranoside (IPTG) (Qiagen,Valencia, Calif.) was then added to a final concentration of 1 mM forthe induction of expression at 25° C. for 6 h. Bacterial cells werecollected by centrifugation, lyzed by Bugbuster (Novagen, Gibbstown,N.J.) and sonicated according to the manufacturer's instructions.Glutathione Sepharose 4B columns were equilibrated with phosphatebuffered saline (PBS, pH 7.4) and lysate supernatant samples were loadedat a flow rate of approximately 1 mL/min. The columns were then washedwith three column-volumes of PBS. Finally, 50 mM Tris-HCl buffer (pH7.4) containing 20 mM glutathione was used to elute the recombinantprotein that was further purified with Mono Q 5/50 GL (GE Healthcare)with 20 mM Tris buffer (pH 6.8). Purified recombinant proteins wereanalyzed by SDS-PAGE (4-15% separation gel). Yields of rhTEM1-GST andrGST proteins were approximately 1 mg and 35 mg per liter of culture,respectively. Recombinant proteins were biotinylated using the EZ-LinkSulfo-NHS-Biotin-Reagents kit according to the manufacturer'sinstructions and dialyzed against PBS.

The full length hTEM1 cDNA was cloned into MluI/PacI-digested lentiviralplasmid vector pHRSIN-GFP to generate pHRSIN-TEM1 vector that expresseshuman TEM1 but not GFP. The corresponding lentivirus was generated bytransient transfection of HEK293T cells with calcium phosphate.Conditioned medium containing viral vectors was harvested 24 and 48hours after transfection, filtered (0.45 μm) and frozen at −80° C. untiluse. MS1, H5V and SKOV3 cells (2×10⁴ per 24-well plate) were transducedusing 500 μL viral supernatants, and expression of hTEM1 was confirmedby RT-PCR (see FIG. 9 and methods), Western blot and flow cytometryanalysis using a rabbit anti-TEM1 polyclonal antibody (kindly providedto GC by Morphotek, Inc, Malvern, Pa.).

Real-Time RT-PCR

Whole cell RNAs were prepared from cells using Trizol (Invitrogen, USA)according to the manufacturer's instructions. Total RNA wasreverse-transcribed using Superscript First-Strand Synthesis Kit forRT-PCR (Invitrogen) under conditions described by the supplier. Primers,TaqMan probes and TaqMan assay reagents for human and mouse TEM1 and 18Swere purchased from Applied Biosystems (ABI, USA). Human and murine TEM1cDNA was quantified by real-time PCR on the ABI Prism 7900 SequenceDetection System (Applied Biosystems, Foster City, Calif.). PCR wasperformed according to manufacturer's instructions using ABI inventoriedprimer probes for human and mouse TEM1 and 18S as endogenous control aswell as Taqman PCR core reagents. Amplification and detection wasperformed under the following conditions: one cycle of 50° C. for 2 min,one cycle of 95° C. for 10 min and 40 cycles each of 95° C. for 15 s and60° C. for 1 min. Post-qRT-PCR calculations to analyze relative geneexpression were performed by the comparative CT method (2^(−ΔΔCT)method). ⁴²Values were normalized with 18S as an endogenous control andMS1 cells was used as a calibrator, set as reference with a value of 1.All other test groups were set as being x-fold difference relative tothe reference.

Flow Cytometry Analysis

Analysis of scFv expression by yeast was performed as previouslydescribed. ⁴ Binding of anti-TEM1 scFvs and biobodies to TEM1-expressermammalian cells was evaluated using various human or murine cell linesexpressing TEM1 endogenously (HEK-293T, MOV1 and 2H11) or stablytransduced with pHRSIN-TEM1 (hTEM1-MS1, hTEM1-H5V and hTEM1-SKOV3). MS1(murine endothelium), H5V (murine endothelium) and SKOV3 (human ovariancancer) were used as negative or weakly positive control cell lines.Anti-TEM1 scFvs were preincubated for 30 min at RT with APC-anti-V5 at aratio 1:1 and anti-TEM1 biobodies were preincubated with APC-labeledstreptavidin at a ratio of 1:4. A non-relevant scFv was used as anegative control for binding.

Shuffling of Phage-Display scFv Library into Yeast

ScFvs initially cloned in the M13 phage display vector pComb3X (ScrippsResearch Institute, La Jolla, Calif.) were rescued by PCR from aphagemid DNA preparation. The primers were designed to bind to theoriginal phage library scFv 5′ and 3′ end sequences, as well as topromote homologous recombination with the yeast-display vector pAGA2.Since the scFv constructs comprised light chain (kappa or lambda)variable regions (V_(L)'s) followed by a GS-rich linker peptide followedby γ heavy chain variable region sequences (V_(H)'s), forward primersannealed to the 5′ ends of V_(L)'s and reverse primers annealed to theconserved 5′ end of the γ_(1,2,3,4)C_(H)1 domain of the IgG heavy chain.

Primer Sequences for scFv Amplification from TTP Phage Library andCloning by Homologous Recombination in pAGA2.

Forward primers: K1-F Ggactggtggtggaggactggtggtggtggatctgtcgacatccagatgacccagtctccatcc (SEQ ID NO: 5) K24-FGgttctggtggtggaggttctggtggtggtggatctgtcga tattgtgatgacycagtctccactc(SEQ ID NO: 6) K3-F ggttctggtggtggaggttctggtggtggtggatctgtcgaaatwgtgwtgacrcagtctccagsc (SEQ ID NO: 7) K5-Fggttctggtggtggaggttctggtggtggtggatctgtcga aacgacactcacgcagtctccagca(SEQ ID NO: 8) Lam1a Ggttctggtggtggaggttctggtggtggtggatctgtccagtctgtgctgactcagccaccctcg (SEQ ID NO: 9) Lam1bggttctggtggtggaggttctggtggtggtggatctgtcca gtctgtgytgacgcagccgccctca(SEQ ID NO: 10) Lam2 ggttctggtggtggaggttctggtggtggtggatctgtccagtctgccctgactcagcctccctcc (SEQ ID NO: 11) Lam 3Ggttctggtggtggaggttctggtggtggtggatctgtctc ctatgagctgactcagccaccctcag(SEQ ID NO: 12) Lam 4 Ggttctggtggtggaggttctggtggtggtggatctgtcctgcctgtgctgactcaatcgccctctg (SEQ ID NO: 13) Lam 6Ggttctggtggtggaggttctggtggtggtggatctgtcaa ttttatgctgactcagccccactctg(SEQ ID NO: 14) Lam 78 Ggttctggtggtggaggttctggtggtggtggatctgtccagactgtggtgacycaggagccmtcac (SEQ ID NO: 15) Lam 9Ggttctggtggtggaggttctggtggtggtggatctgtcca gcctgtgctgactcagccaccttctg(SEQ ID NO: 16) Lam 10 Ggttctggtggtggaggttctggtggtggtggatctgtccaggcagggcagactcagcagctctcgg (SEQ ID NO: 17) Reverse primer: 1234-Bgtcttcttcagaaataagcttttgttcggatccctcgaact ggccactagtgaccgatgg(SEQ ID NO: 18)

PCR conditions for scFv amplification were: 94° C. for 5 min followed by25 cycles of 94° C. 1 min, 55° C. 1 min and 72° C. 1 min, and a finalextension of 7 min at 72° C. PCR products were then purified byelectrophoresis using the Qiaquick Gel Extraction Kit (Invitrogen,Carlsbad, Calif.). pAGA2 vector for yeast display was linearized withNheI and XhoI and purified using the Qiaquick PCR Purification Kit(Invitrogen, Carlsbad, Calif.). EBY100 competent cells were prepared forelectroporation according to the condition G (Table 2), andco-transfected with purified PCR products and pAGA2 linearized vector.Transfected yeast cells were finally expanded in SD-CAA medium at 30° C.at 200 rpm until saturation. Ten-fold serial dilution of the transfectedyeast were cultured on SD-CAA plates for calculation of the library'ssize.

The library's diversity and gap repair efficiency were evaluated bysequencing and flow cytometry analysis, respectively. In brief, 30individual clones were randomly selected, induced in the mediumSGRD-CAA, and assessed for scFv expression using the c-myc tag expressedonly by yeast displaying scFv. Plasmid DNA was extracted from theseclones using MasterPure Yeast DNA Purification Kit and scFv genefragments were amplified for sequencing with primers flanking the gaprepair sites. PCR amplification primers are: pAGA2-scFv-For:5′-ccgtactctttgtcaacgac-3′(SEQ ID NO: 41) and pAGA2-scFv-Rev:5′-ttaaagccttcgagcgtccc-3′(SEQ ID NO: 42) Sequencing primers are:pAGA2-seq-For: 5′-gggaaggcaatgcaagga g-3′ (SEQ ID NO:19) andpAGA2-seq-Rev: 5′-tgcgtacacgcgtctgtacag-3′ (SEQ ID NO: 20).

Antibodies

Yeast-display scFv expression was detected with anti-c-myc mousemonoclonal antibody (mAb), 9E10 (Santa Cruz Biotechnology, Inc., SantaCruz, Calif.) and Alexa488 F(ab′)2 fragment of goat anti-mouse IgG (H+L)(488 anti-IgG) (Invitrogen, Carlsbad, Calif.) or PE-Cy5 goat F(ab′)2anti-mouse IgG (H+L) (PE-Cu5 anti-IgG) (Cedarlane Laboratories Limited,Burlington, N.C.). Biotinylated antigen binding to yeast-display scFvwas detected with goat anti-biotin-FITC (Abcam, Cambridge, Mass.) orstreptavidin-PE (BD Pharmingen, San Jose, Calif.). ScFv binding to celllines were detected with APC-conjugated anti-V5 mouse mAb (AbD Serotec,Raleigh, N.C.) (APC anti-V5) and scFv binding to plastic-immobilizedantigen was detected by HRP-conjugated mouse anti-V5 mAb (AbD Serotec)(HRP-anti-V5). Biobody binding to TEM1-expresser cells was detected withAPC-labeled streptavidin (eBioscience, San Diego, Calif.). Confocalmicroscopy was performed with anti-SV40 Tag mAb (Santa-Cruz Biotech,Santa Cruz, Calif.) detected by goat anti-mouse IgG1k-Alexa 488 mAb (488anti-IgG1k) (Invitrogen, Carlsbad, Calif.), and rhodamine-labeledstreptavidin (Invitrogen).

Identification of anti-TEM1 scFv

The TTP yeast-display scFv library was first screened for anti-TEM1 scFvby magnetic and flow sorting as described. Anti-TEM1 yeast-display scFvswere then transformed into yeast-secreted scFv and screened by detectionELISA assays. Briefly, the library was magnetically enriched once forscFvs that bound to 20 nM of biotinylated rhTEM1-GST, twice for scFvsthat bound to 6 nM of biotinylated rhTEM1-GST, then magneticallydepleted for scFv that bound to 60 nM biotinylated rGST. Anti-TEM1yeast-display scFvs were then flow sorted for c-myc/TEM1 double positiveclones. Progressively decreasing concentrations of rhTEM1-GST were usedduring the screening (from 2 nM to 400 pM). DNA plasmids were extractedfrom yeast-display scFv that bound to 400 pM of rhTEM1-GST but not to 2nM of rGST, and scFv fragments were amplified using primers allowinghomologous recombination with the secretion vector p416-BCCP. Theprimers used were: Forward shuffling primer:5′-ggttctggtggtggaggttctggtggtggtggatct g-3′(SEQ ID NO: 21); Reverseshuffling primer:5′-gagaccgaggagagggttagggataggcttaccgtcgaccaagtcttcttcagaaataagctt-3′(SEQ ID NO: 22). ScFv fragments and linearized p416-BCCP wereco-transfected into YVH10 by chemical transformation. Soluble scFvscreening for specific binding to TEM1 was performed by ELISA with yeastsupernatants of 470 random transformants in Maxisorp ELISA plates (Nunc,Rochester, N.Y.) coated with 0.8 μg/ml of rhTEM1-GST vs. rGST.Recombinant proteins were plastic-immobilized in carbonate-bicarbonatebuffer 0.5 M, pH 9.6 (Fisher Scientific, Pittsburgh, Pa.) overnight (ON)at 4° C. Wells were then blocked with 5% dry milk in PBS (PBMS) (Biorad,Hercules, Calif.) for 2 hours at RT with gentle agitation and incubatedwith yeast supernatants diluted 1:1 with PBSM at RT for 1 h. After threewashes with PBS supplemented with 0.05% Tween (PBST) (Biorad), scFvbinding to immobilized proteins was detected with HRP anti-V5.Colorimetric signals were developed with TMB substrate solution (KPL,Inc., Gaithersburg, Md.), quenched with sulfuric acid (KPL, Inc) andread at 450 nm on a Fluoroskan Ascent FL (Thermo Fisher Scientific,Pittsburgh, Pa.). Approximately 50% of the colonies (232/470) gave acolorimetric signal higher than the average background plus 3 standarddeviations. No cross-reactivity with rGST control protein was detected.Sequencing of 40 scFv identified five unique clones that were thenproduced and Ni-purified as previously described.

Measurement of scFv Affinity by ELISA

To assess scFv-78 affinity maxisorp ELISA plates were coated withrhTEM1-GST at two-fold decreasing concentrations from 0.4 to 0.05 μg/ml,in carbonate-bicarbonate buffer. After blocking with PBSM, wells wereincubated with ten-fold serial dilutions of scFv-78, starting from 1 μM.ScFv binding to immobilized proteins was detected with HRP-conjugatedmouse anti-V5 monoclonal antibody. Colorimetric signals were developedas previously described. The same procedure was followed for the otherscFvs, but using 2 and 1 μg/ml of coated rhTEM1-GST and three-foldserial dilutions of scFv, starting at 2 μM.

Orthotopic Mouse Model of Ovarian Cancer

MOV-1 mouse ovarian cancer cell line was derived from an ovarian cancerthat spontaneously arose in Tg-MISIIR-TAg mice. MOV-1 cell lineexpresses SV40 antigen and TEM1. To emulate ovarian cancer mouse ovariancancer cells, MOV1 were orthotopically injected in the ovarian bursa ofNOD-Scid-γc^(null) (NSG) mice. Four month-old females were anesthetizedaccording to the protocol approved by the University of PennsylvaniaInstitutional Animal Care and Use Committee (IACUC). A dorso-lateralincision on right caudal portion of the animal dorsum was made. Theretroperitoneum was dissected to expose the left ovary using the forcepsto grasp, retract, position, and secure the organ for injection. Fivemillion MOV1 cells were injected in the ovarian bursa in a volume of 20ml of PBS using an insulin syringe. Retroperitoneum wounds were closed,animals were administered antibiotics and fluids, and tumor growth wasmonitored by in vivo imaging.

Analysis of In Vivo Distribution of Anti-TEM1 Biobodies by ConfocalMicroscopy

Anti-TEM1 biobodies were injected IV 3 weeks after tumor cellimplantation. Spleen, liver, kidney and ovaries were harvested 24 or 48hours after biobody injection and preserved in frozen tissue matrix OCTcompound (Tissue-Tek, Sakura Finetek USA). Slides of 5 mm thickness werecut from frozen sections, air dried 1 hr at RT and fixed by immersion incold 100% acetone 5 min. After 2 washes in PBS, slides were blocked forendogenous biotin by pre-treatment with Avidin/Biotin Blocking solution(avidin-skim milk 0.001% in PBS). Biobody binding was detected withrhodamine-conjugated streptavidin. Tumor cells were detected with 2μg/ml of SV40 Tag antibody for 30 min at RT followed by 1 μg/ml Alexa488 goat anti-mouse IgG1κ for 30 min at RT. Slides were incubated with1:2000 diluted DAPI (Invitrogen) for 30 min at RT to visualize thenuclei. Fluorescent signals were acquired by confocal analysis (ZeissLSM 510META NLO) at 63× magnification

Epitope Mapping of scFv78 on Human TEM1

In order to further characterize scFv78, epitope mapping was performedusing yeast display-peptides. The advantages of yeast-display peptidesover synthesized peptides include 1) presence of post-translationmodifications; 2) no need of purification; 3) entirely renewable; 4)cost-effectiveness. Our novel vector pAGA2 designed for yeast displayenables cloning by homologous recombination and c-mcy tagging of theyeast-display peptides.

Six overlapping fragments of 200-220 bp were designed to cover theN-terminal extracellular domain of TEM1 (1.2 kb) that was used toisolate the recombinant antibody scFv78 from our novel yeast-displayscFv library. The fragments were amplified by PCR and ligated byhomologous recombination into pAGA2 vector. Epitope mapping wasperformed by flow cytometry biotinylated scFv78 (biobody) to probe theyeast-display TEM1-peptides. scFv78 biobody bound only to T6 peptide.The findings indicate that the binding site of scFv78 is situatedbetween 324aa and 390aa.

Primers to amplify the small TEM1 fragments T1 (20-88aa), T2 (78-152aa),T3 (142-216aa), T4 (206-275aa), T5 (265-334aa) and T6 (324-390aa) areshown below according to the Genebank Access Number: NM_020404. Genefragments of each small peptide were amplified by PCR and cloned intothe yeast display vector pAGA2 via gap repair (homologous recombination)by co-transformation of the PCR fragments and the vector into yeaststrain EBY100. Colonies with right gene insertion were induced todisplay the peptides on yeast surface and scFv78 biobody binding wasevaluated by FACS using PE conjugated streptavidin.

Primers for the six small peptides cloning:

T1F: (SEQ ID NO: 23) Ggt ggt gga ggt tct ggt ggt ggt gga tct Ccctgggctgctgagccc T1R: (SEQ ID NO: 24)TTCTTCAGAAATAAGCTTTTGTTCGGATCCCTGCAGCCCGATCCACAG T2F: (SEQ ID NO: 25)Ggt ggt gga ggt tct ggt ggt ggt gga tct ccagccagcc ggctgctg T2R:(SEQ ID NO: 26) TTCTTCAGAAATAAGCTTTTGTTCGGATCCGTCGACAGCCAGCGTGCAC T3F:(SEQ ID NO: 27) Ggt ggt gga ggt tct ggt ggt ggt gga tct tggctggagggctcgtgc T3R: (SEQ ID NO: 28)TTCTTCAGAAATAAGCTTTTGTTCGGATCCCTGCTTCACGCAGAGCAGAG T4F: (SEQ ID NO: 29)Ggt ggt gga ggt tct ggt ggt ggt gga tct ggcagggga gcctctctg T4R:(SEQ ID NO: 30) TTCTTCAGAAATAAGCTTTTGTTCGGATCCACAGGGGTCCTCGCAACTG T5F:(SEQ ID NO: 31) Ggt ggt gga ggt tct ggt ggt ggt gga tct gcagcagacgggcgcagttg T5R: (SEQ ID NO: 32)TTCTTCAGAAATAAGCTTTTGTTCGGATCCCTCGAAGCCACCAACGTAG T6F: (SEQ ID NO: 33)Ggt ggt gga ggt tct ggt ggt ggt gga tct cagatgtgtg tcaactacgttgg T6R:(SEQ ID NO: 34) TTCTTCAGAAATAAGCTTTTGTTCGGATCCCGTCCAGCCACCGTTGAAG

Amino acids sequences of the six small peptides:

T1 (20-88aa): (SEQ ID NO: 35)P W A A E P R A A C G P S S C Y A L F P R R R T F LE A W R A C R E L G G D L A T P R T P E E A Q R V DS L V G A G P A S R L L W I G L Q T2 (78-152aa): (SEQ ID NO: 36)P A S R L L W I G L Q R Q A R Q C Q L Q R P L R G FT W T T G D Q D T A F T N W A Q P A S G G P C P A QR C V A L E A S G E H R W L E G S C T L A V D T3 (142-216aa):(SEQ ID NO: 37) W L E G S C T L A V D G Y L C Q F G F E G A C P A LQ D E A G Q A G P A V Y T T P F H L V S T E F E W LP F G S V A A V Q C Q A G R G A S L L C V K Q T4 (206-275aa):(SEQ ID NO: 38) G R G A S L L C V K Q P E G G V G W S R A G P L C LG T G C S P D N G G C E H E C V E E V D G H V S C RC T E G F R L A A D G R S C E D P C T5 (265-334aa): (SEQ ID NO: 39)A A D G R S C E D P C A Q A P C E Q Q C E P G G P QG Y S C H C R L G F R P A E D D P H R C V D T D E CQ I A G V C Q Q M C V N Y V G G F E T6 (324-390aa): (SEQ ID NO: 40)Q M C V N Y V G G F E C Y C S E G H E L E A D G I SC S P A G A M G A Q A S Q D L G D E L L D D G E D EE D E D E A W K A F N G G W TResults

Example 1 Generation of Vectors for Yeast-Display And Yeast-Secretion ofN-Terminal Biotinylated scFv

To overcome the loss of antigen specificity and/or affinity due to scFvtransfer from cell surface display to secreted, two complementaryvectors were developed, pAGA2 and p416 BCCP, which permit scFv to bedisplayed (FIG. 1a ) or secreted (FIG. 1b ) by the same expressionsystem (S. cerevisiae) and through engineering at the same domain (Nterminus), thus with similar post-translational modifications andconformation. In pAGA2 vector, scFv are fused at the N-terminus to Aga2,to permit display (FIG. 1). ScFv expressed by p416 BCCP are soluble andfused at the N-terminus to an enzymatically biotinylable domain (BCCP)separated from the scFv functional site by a flexible IgA hinge (FIG.1). The presence of the IgA hinge minimizes conformational effects onscFv by biotinylated BCCP during its binding to immobilized or labeledstreptavidin. The BCCP domain can be specifically biotinylated in vitrowith a recombinant biotin ligase. To achieve in vivo biotinylation andsecretion of biotinylated scFv (biobodies), mating of yeast-secretingscFv with yeast bearing biotin ligase has been developed. The vectorspAGA2 (FIG. 1c ) and p416 BCCP (FIG. 1d ) were derived from the shuttlevectors p414 GAL1 and p416 GAL1, allowing galactose-inducible expressionin the presence of uracil or tryptophane, respectively, which minimizesgrowth bias.

Example 2 Optimization of Yeast Transformation

Although chemical transformation with lithium acetate (LiAc) orelectroporation can be used for yeast display library construction, itremains to date unclear whether these methods differ in transformationefficiency, especially for the strains used to display or secrete scFv(EBY100 and YVH10, respectively). Reported here is the firstoptimization of both methods. Table 1 shows the procedure for chemicalcompetent cell preparation and transformation. Optimization wasconducted in two steps, first by calibrating competent cell preparationand transformation, and then by optimizing DNA input. Condition 2 wasbest for chemical transformation (Table 1 and FIG. 2a ) and was appliedfor the transformation of both EBV100 and YVH10 stains with intactplasmid or with combinations of linearized vector and insert forhomologous combination (FIG. 2b ). YVH10 chemical transformation yielded10⁶ transformants per microgram of DNA by homologous recombination,which was unexpectedly higher than transformation with intact plasmid(FIG. 2b , gray bars). However, for EBY100 yeast the chemicaltransformation efficiency was low (FIG. 2b , black bars). Thus, wedeveloped an alternate protocol for EBY100 transformation usingelectroporation (Table 2). While electroporation of YVH10 resulted inlow yield and was not pursued (FIG. 3), condition G (Table 2 and FIG. 2c) gave the highest yield of transformants for EBV100, producing 1×10⁸transformants per electroporation with 10 μg of linearized vector andinsert combined (FIG. 2d ). Thus, both YVH10 and EBV100 yeast strainsare more efficiently transformed by homologous recombination than byintact plasmids. YVH10 is best transformed by chemical transformationwith heat shock, while EBV100 is best transformed by electroporation,and at levels compatible with generation of scFv libraries.

TABLE 1 Yeast Yeast washes Incubation Heat Yeast Condi- Yeast w/water &w/vector + Shock recovery tions resuspension LiAc/TE insert at 42° C. InYPED 1 Chill in ice 4° C. w/o DMSO 30 min 2 h 2 Room Temp. 4° C. w/oDMSO 30 min 2 h 3 Room Temp. 4° C. w/DMSO 30 min 2 h 4 Room Temp. RT w/oDMSO 30 min 2 h 5 Room Temp. RT w/DMSO 15 min 2 h

TABLE 2 shaking conditions [DTT] buffer [sorbitol] time shaking T° A 25mM 15 min 30° C. B 25 mM 30 min 30° C. C 25 mM 10 mM Tris 30 min 30° C.D 25 mM 20 mM Hepes 30 min 30° C. E 25 mM 1M 30 min 30° C. F 25 mM 1M 60min 4° C. G 25 mM 20 mM Hepes 0.6M  30 min 30° C. H 25 mM 20 mM Hepes 1M30 min 30° C. I 25 mM LiAc-TE 0.6M  30 min 30° C. J 25 mM LiAc-TE 0.6M 60 min 4° C. K 25 mM LiAc-TE 1M 30 min 30° C. L 25 mM LiAc-TE 1M 60 min4° C. M 25 mM LiAc-TE 30 min 30° C. N 25 mM LiAc-TE 30 min 4° C. O 25 mMLiAc-TE 60 min 4° C.

Example 3 Construction and Validation of TTP Yeast-Display scFv Library

The M13 phage-display human scFv library derived from a patient withthrombotic thrombocytopenic purpura (TTP), a coagulation system disordercaused by autoantibodies to the metalloprotease ADAMTS13, was previouslyreported. ScFv gene segments from the TTP phage display library wererescued by PCR from a phagemid DNA preparation, and co-transformed withpAGA2 linearized vector by electroporation in EBV100 yeast strain toallow homologous recombination and cell surface display. The diversityof the resulting yeast-display scFv library was validated by thesequencing of 20 randomly selected clones that demonstrated a gap repairrate of 95% (data not shown). Yeast-display scFv was assessed by flowcytometry through the detection of c-myc tag that is fused to the scFvC-terminus. Nine out of 24 randomly selected clones displayed scFvs ontheir surface after induction (FIG. 4 clones a,g,i,k,o,p,r,u,w), whichwas consistent with the expression ratio of phage and yeast librariespreviously reported.

Example 4 Identification of Anti-TEM1 scFv

TEM1 or endosialin was first described as a marker of tumor endothelialcells. Other studies showed that endosialin is highly expressed onpericytes and stroma cells related to tumor angiogenesis, proliferation,migration and metastasis. Recently, endosialin was described as both atumor endothelial and an endothelial precursor cell marker with highexpression in tumors and little or no expression in normal tissues.Therefore, targeting TEM1 with antibody or antibody-conjugated reagentssuch as an immunotoxin, isotope, or nanoparticles is a promisingapproach for both diagnosis and therapy. The TTP yeast-display librarywas enriched for scFv that bind to recombinant human TEM1-GST protein(rhTEM1-GST) first by magnetic and then by flow sortings and wasdepleted by magnetic sortings from scFv binding to control recombinantGST (rGST) protein, as previously described. Magnetic sorting is basedon one parameter of selection only (capture of antigen-binding yeast),thereby providing a rapid means for robust enrichment ofantigen-specific scFv. Flow sorting uses two parameters of selection,including antigen binding and recognition of c-myc tag on scFv displayedat the yeast cell surface, thereby further enriching forantigen-specific scFv while preventing the selection of yeast that bindto antigen directly through their coat. DNA was extracted fromdouble-positive sorted yeast to amplify scFv sequences with primersallowing homologous recombination with p416 BCCP vector. Resultingtransformants secreted scFv enriched for TEM1-specific binding.

Four hundred and seventy yeast-secreting scFv were tested by ELISA fortheir ability to detect rhTEM1-GST. Almost 50% (232/470) of the solublescFv bound to rhTEM1-GST but not to rGST. Anti-TEM1 soluble scFv wereclassified into two affinity categories: “high” when the optical density(OD) by detection ELISA was greater than 0.5, and “low” when lower than0.5. Twenty scFv of each category were sequenced. The high OD groupcontained only one sequence (scFv-78) and three clones presented eachone point mutation in the VL; #6 had a point mutation in FR1 thatchanged a serine in leucin; #8 a point mutation in FR3 changing a glycinin valine; #10 a point mutation in CDR2 changing a leucine inmethionine. The low OD group included four different scFvs (scFv-131;scFv-132; scFv-133; scFv-137) (FIG. 5). Alignment of scFv heavy andlight chain variable region sequences to a database of humanimmunoglobulin germline sequences (V Base Directory of Human V GeneSequences) indicated that the heavy chain variable region sequences usedVH3- and VH4-family-encoded gene products and showed extensive somaticmutation particularly in the CDR regions as though they evolved duringan antigen-driven immune response (FIG. 5a ). The light chain sequencesused by all 5 anti-TEM1 scFv's were lambda and were minimally mutatedfrom their most likely VL and JL germline genes (FIG. 5b ).

Example 5 Characterization and In Vitro Validation of Anti-TEM1 scFvSand Biobodies

ELISA provides a convenient way to evaluate the affinity of an antibody.Because a scFv has only one binding site for antigen, the affinity canbe calculated by the equation of Kd=2[Ab′]_(t)-[Ab]_(t), derived fromthe Beatty's equation K_(aff)=1/2(2[Ab′]_(t)-[Ab]_(t)), where [Ab′]_(t)refers to scFv concentration at half the maximal OD (OD₅₀) forrhTEM1-GST-coated wells at half concentration, and [Ab] refers to scFvconcentration at OD₅₀ for rhTEM1-GST-coated wells at wholeconcentration. The calculated Kd of the five anti-TEM1 scFvs withdistinct sequences were 4.3+/−1.5 nM for scFv-78 (FIG. 6), 148 nM forscFv-132, 218 nM for scFv-133, 682 nM for scFv-131, and 4.4 μM forscFv-137, respectively (FIG. 7). R² of the curve fittings for all ELISAswere above 0.99. ScFv were then biotinylated on the BCCP site inN-termini by yeast mating with biotin ligase-bearing yeast to producebiobodies, as described by Scholler et al., and expressed at a yield of10 mg per liter.

Flow cytometry showed that all anti-TEM1 scFvs bound to the humanTEM1-transfected mouse endothelial cell line hTEM1-MS1, but not towild-type MS1 cells, and binding could be blocked with rhTEM1 but notwith rGST (FIG. 8a-l ). The remainder of the study was performed withthe high affinity anti-TEM1 scFv-78 and low affinity scFv-137 aftertargeted biotinylation, resulting in biobody-78 and biobody-137,respectively. Biobody-78 strongly bound to cell lines transduced withhuman TEM1 (FIG. 8.o, q) and cells expressing high levels of endogenoushuman (FIG. 8.m) or mouse (FIG. 8.r, s) TEM1. Biobody-78 also bound tocell lines that express moderate levels of endogenous mouse or humanTEM1, such as H5V (FIG. 8.n) and SKOV3 (FIG. 8.p), respectively. TEM1mRNA expression level was verified by qRT-PCR in all cell lines (FIG.9).

Example 6 In Vivo Validation of Anti-TEM1 scFVs

To test the ability of anti-TEM1 scFvs to recognize target in vivo,immunodeficient mice were transplanted orthotopically in the leftovarian bursae with MOV1 ovarian cancer cells, which express mouse TEM1(FIG. 9B). Groups of mice were injected IV with a bolus of either thehigh-affinity biobody-78 or the low affinity biobody-137 (50 μg/mouse).Animals were euthanized after 24 or 48 hours, and spleens, livers,kidneys and ovaries were collected to monitor biobody distribution byconfocal microscopy. Twenty-four hours after IV injection, anti-TEM1biobody-78 was detected in kidneys but was cleared after 48 hours. Bothanti-TEM1 biobody-78 and biobody-137 specifically localized to MOV1cells implanted in the left ovaries after 48 hours but not in thecontralateral normal ovaries or in the other normal mouse organs (FIG.10).

Example 7 Epitope Mapping

scFv78 biobody binds specifically onto the yeast displaying the small T6peptide (324-390aa) and the control big N terminal peptide (20-390aa)(FIG. 11). The epitope on human TEM1 for scFv78 targeting is 324-390amino acids residue, located in the middle of the extracellular domain(18-687aa).

Example 8 Internalization of Anti-TEM1 Recombinant Antibody

To assess the specific internalization of anti-TEM1 recombinant antibodyin TEM1-expresser cells, we used two endothelial cell lines, theparental MS1 cell line and the TEM1-transduced MS1-TEM1 cell line. Afterfixation and permeabilization, cells were incubated in complete mediumat 37° C. for 15 h with anti-TEM1 scFv78 detected by a fluorescentlyconjugated anti-V5 antibody (FIG. 12A,D) or with the site-specificbiotinylated anti-TEM1 biobody78 bound to fluorescent beads (FIG.12F,H). Fluorescent beads were obtained by incubatingstreptavidin-coated magnetic beads (Miltenyi) with a fluorescentconjugate (dylight648-labeled and biotinylated BSA). Confocal imagingdemonstrated the specific internalization of both anti-TEM1 scFv78 andbiobody78 in MS1-TEM1 cells (FIG. 12D, H), but not in wild type MS1(FIG. A, F). As negative controls, MS1 cells were incubated withBSA-coated fluorescent beads (FIG. 12E), and MS1-TEM1 cells wereincubated in medium (FIG. 12B), with B7H4-V5 tag (FIG. 12C), orBSA-coated fluorescent beads (FIG. 12G).

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications that are within the spirit and scopeof the invention, as defined by the appended claims.

What is claimed is:
 1. A method of diagnosing the presence of a tumor ora cancer growth in a subject, said method comprising: (a) isolating atissue sample from said subject; and (b) sampling said tissue sampleisolated from said subject with a composition comprising an antibody orantigen-binding fragment thereof, wherein said antibody orantigen-binding fragment is specific for both the mouse and human formof an endosialin tumor endothelial marker 1 (TEM1) and binds to anepitope sequence as set forth in SEQ ID NO: 40, wherein the antibody orantigen-binding fragment comprises a heavy chain variable regioncomprising a CDR1 sequence of residues 31 to 35 of SEQ ID NO: 43; a CDR2sequence of residues 50 to 65 of SEQ ID NO: 43; and a CDR3 sequence ofresidues 98 to 102 of SEQ ID NO: 43, and wherein the antibody orantigen-binding fragment comprises a light chain variable regioncomprising a CDR1 sequence of residues 23 to 36 of SEQ ID NO: 48; a CDR2sequence of residues 52 to 62 of SEQ ID NO: 48; and a CDR3 sequence ofresidues 97 to 104 of SEQ ID NO: 48, whereby specific binding of saidantibody or antigen-binding fragment to said tissue sample is indicativeof the presence of a tumor or cancer growth in said subject.
 2. Themethod of claim 1, further comprising a secondary reagent thatspecifically binds to said antibody or antigen-binding fragment but doesnot antagonize binding of said antibody or antigen-binding fragment toits target.
 3. The method of claim 2, whereby said secondary reagent isa photoactivatable agent, a fluorophore, a radioisotope, abioluminescent protein, a bioluminescent peptide, a fluorescent tag, afluorescent protein, or a fluorescent peptide.
 4. The method of claim 1,whereby said sampling comprises the step of analyzing said sample usinga chromogenic immunological assay.
 5. The method of claim 4, wherebysaid sampling comprises the step of analyzing said sample usingmicroscopic imaging.